THE  PROPERTY  OF 

!ir?i  One  if  ft?  Pacific. 


MEMCAL    SCHOOL 


MANUAL 


OF 


BACTERIOLOGY 


Plate  L 


BACTERIA,  SCHIZOMYCETES    OR  FISSION- FUNGI. 

wi  Tc  del.  VincentBroote.Day  &Son. 


MANUAL 


OF 


BACTERIOLOGY 


BY 


EDGAR     M.     CROOKSHANK 
^ 

M.B.  (LOND.),  F.R.M.S. 

DEMONSTRATOR    OF     PHYSIOLOGY,     KING'S    COLLEGE,     LONDON 


SECOND  EDITION,   REVISED  AND  CONSIDERABLY  ENLARGED 
ILLUSTRATED  WITH  COLOURED  PLATES  AND  WOOD  ENGRAVINGS 


J.  H.  VAIL  AND  CO. 
21,  ASTOR  PLACE,  NEW  YORK 

,1,88?        ,,,      ,»       'I.. 


iVTb 


SIR    JOSEPH    LISTER,    BART.,    M.B.,    F.R.S., 

WHO   HAS   CREATED  A  NEW  EPOCH   IN 

MEDICINE  AND  SURGERY, 

BY  APPLYING  A  KNOWLEDGE  OF  MICRO-ORGANISMS 
TO  THE  TREATMENT  OF  DISEASE, 

TO0  CHorit  i*  DetucateD 

AS  A 
TOKEN  OF  ADMIRATION  AND  RESPECT 

BY   THE   AUTHOR. 


13561 


PREFACE 

TO    THE    SECOND    EDITION, 


THE  fact  that  a  new  edition  of  this  manual  was 
called  for  a  few  months  after  its  publication,  has 
induced  the  author  to  extend  its  scope  in  the 
hope  of  adding  to  its  usefulness. 

The  work  has  not  only  been  revised  throughout 
and  brought  up  to  date,  but,  in  order  to  admit 
of  a  more  concise  arrangement  of  the  species,  the 
Systematic  part  has  been  recast. 

Additional  chapters  have  been  written  upon  the 
General  Morphology  and  Physiology  of  Bacteria, 
upon  Antiseptics  and  Disinfectants,  and  Immunity. 

Seventy-three  illustrations  have  been  added. 
Those  not  duly  acknowledged  as  coming  from 
other  sources  were  drawn  on  the  wood  by  the 
author  from  his  own  preparations. 

A  list  of  references  to  works  on  Bacteriology, 
which  was  not  ready  for  the  first  edition,  has 
now  been  completed  and  extended.  It  has  no 
pretension  to  be  a  complete  bibliography,  but 
being  arranged  as  much  as  possible  in  accordance 


Vlll  PREFACE    TO    THE    SECOND    EDITION. 

with  the  chapters,  and  in  chronological  order,  may 
be  useful  to  those  seeking  further  details.  No 
doubt  Professor  Baumgarten's  Jahresbericht,  the 
first  number  of  which  has  been  issued  this  year, 
will  be  found  a  valuable  guide  to  current  literature 
in  the  future. 

The  author  desires  again  to  express  his  ac- 
knowledgments to  Professor  Gerald  Yeo  and 
Mr.  Herroun,  of  King's  College,  London. 

EDGAR   M.    CROOKSHANK. 


24,  MANCHESTER  SQUARE,  W., 

December,  1886. 


PREFACE 

TO    THE    FIRST    EDITION. 


A  BRANCH  of  study,  which  has  opened  fresh 
paths  in  pathology  and  therapeutics,  is  so 
important  that  it  may  in  time  become  a  more 
essential  part  of  the  medical  curriculum,  and  is 
perhaps  worthy  of  an  elementary  handbook,  which 
shall  include  a  systematic  sketch  of  the  genera  and 
species  of  micro-organisms,  as  well  as  the  methods 
employed  in  the  investigation  of  their  life-histories. 

Having  myself  experienced  the  want  of  a  practical 
aid  to  the  study  of  Bacteriology  by  the  methods 
introduced  by  Professor  Koch,  I  thought  that  it 
might  be  useful  to  those  wishing  to  commence  this 
study  by  these  recent  methods,  if  I  embodied  my 
notes  made  in  different  laboratories  in  the  form  of  a 
Manual  for  Students.  The  work  is  thus  intended 
to  help  the  student  beginning  the  study  of  a  subject, 
the  literature  of  which,  in  English,  is  for  the  most 
part  diffused  in  numerous  periodicals. 

The  methods  of  "pure  cultivation"  of  Bacteria 
will,  after  all,  be  found  to  be  remarkable  for  their 
extreme  simplicity,  and  can  be  easily  mastered  by 


X  PREFACE    TO    THE    FIRST    EDITION. 

the  careful  clinical  worker,  to  whom  it  is  hoped  this 
little  book  may  also  prove  useful. 

I  take  this  opportunity  of  expressing  my  best 
thanks  to  Professor  Virchow,  who  materially  fur- 
thered my  work  in  the  Pathological  Institute  of 
Berlin  by  kind  advice  and  generous  assistance. 

I  am  most  grateful  to  Dr.  Babes,  of  Budapest, 
for  his  ever-ready  co-operation ;  and  to  Professor 
Johne,  of  Dresden,  who  also  placed  his  laboratory 
at  my  disposal,  and  to  whom  I  am  particularly 
indebted  for  much  of  the  material  from  which 
the  microscopical  preparations  were  made. 

I  would  also  wish  gratefully  to  acknowledge  the 
great  interest  and  courteous  assistance  shown  me 
on  the  part  of  Dr.  Hauser,  of  Erlangen ;  Professor 
Pettenkofer,  Professor  Bollinger,  and  Dr.  Biichner, 
of  Munich ;  and  the  officials  of  the  new  Hygienic 
Laboratory  in  Berlin. 

The  original  drawings  from  which  the  coloured 
plates  of  test-tube-  and  potato  cultivations  are 
reproduced  were  made  by  my  wife  from  cultiva- 
tions prepared  especially  for  the  purpose,  and 
selected  as  typical.  The  coloured  plates  of  the 
microscopic  appearances  are  reproductions  of  my 
own  drawings,  from  specimens  I  had  recently 
prepared.  The  drawings  were  made  from  parts 
selected  as  most  characteristic  from  a  great  number 


PREFACE    TO    THE    FIRST    EDITION.  XI 

of  preparations,  which  have  been  since  demon- 
strated at  the  meeting  of  the  Royal  Microscopical 
Society,  on  November  25th. 

All  the  plates  have  been  most  faithfully  and 
skilfully  reproduced  by  Messrs.  Vincent  Brooks, 
Day,  &  Son. 

In  conclusion,  I  owe  much  to  Professor  Gerald 
F.  Yeo,  of  King's  College,  London,  for  many 
valuable  criticisms;  and  to  my  colleague,  Mr. 
Herroun,  for  his  kindness  in  reading  the  proof- 
sheets. 

EDGAR  M.  CROOKSHANK. 

24,  MANCHESTER  SQUARE,  W., 
December,  1885. 


CONTENTS. 

PART    I . 

GENERAL  METHODS. 


CHAPTER  I. 

PAGE 

INTRODUCTORY  \ 


CHAPTER  II. 

APPARATUS,  MATERIAL,  AND  REAGENTS  EMPLOYED 

IN  A  BACTERIOLOGICAL  LABORATORY .  .  .        5 

a.  Histological  apparatus          ......         5 

b.  Reagents  and  materials  employed  in  the  processes  of  hardening. 

decalcifying,  embedding,  fixing,  and  cutting  of  tissues .  .         7 

c.  Reagents  for  examining  and  staining  microscopical  preparations  .         9 

d.  Reagents  for  mounting  and  preserving  preparations           .  .16 

e.  Drawing  and  photographic  apparatus           .             .             .  17 

f.  Sterilisation  apparatus  .  .  .  .  .  19 

g.  Apparatus  and    material  for  preparing  and  storing  gelatine-   and 

agar-agar-peptone-broth               .             .             .             .  .21 

h.  Apparatus  for  employment  of  nutrient  jelly  in  test-tube  and  plate 

cultivations          .             .            .             .            .            .  .24 

i.  Apparatus  for  preparation  of  potato  cultivations     .            .  .28 

j.  Apparatus  for  preparation  of  solidified  sterile  blood  serum  .       29 

k.  Apparatus  for  storing,  and  for  cultivations  in,  liquid  media  .       31 

/.  Apparatus  for  incubation      ...                          .  .        32 

m.  Inoculating  and  dissecting  instruments  and  apparatus  in  common  use      41 

n.  General  laboratory  requisites            .            .             .             .  .42 


XIV  CONTENTS. 


CHAPTER  III. 

PAGE 

MICROSCOPICAL  EXAMINATION  OF  BACTERIA  IN 
LIQUIDS,  IN  CULTIVATIONS  ON  SOLID  MEDIA,  AND 
IN  TISSUES .45 

a.  Examination  in  the  fresh  state         .  .  .  .  .46 

b.  Cover-glass  preparations  ;  Methods  of  Ehrlich,  Babes,  and  His   .       48 

c.  Cover-glass  impressions        .  .  .  .  .  52 


CHAPTER  IV. 
FREPARA  TION  AND  STAINING  OF  TISSUE  SECTIONS     .       54 

a.  Methods  of  hardening  and  decalcifying  preparations  .  .       54 

b.  Methods  of  embedding,  fixing,  and  cutting  .  .  -55 

c.  General  principles  of  staining  bacteria  in  tissue  sections  :  methods 

of  Weigert,  Gram,  and  Weigert-Ehrlich  .  .  -57 


CHAPTER  V. 

PREPARATION  OF  NUTRIENT  MEDIA    AND  METHODS 

OF  CULTIVATION         .  .  .  .  .  .62 

SOLID  MEDIA  : 

a.  Preparation  of  sterile  gelatine-,  and  agar-agar-peptone-broth          .       64 

b.  Methods  of  employing  nutrient  jelly  in  test-tube-  and  plate-cultiva- 

tions       ........       69 

c.  Preparation  and  employment  of  sterilised  potatoes,  potato-paste, 

bread-paste,  vegetables,  fruit,  and  white  of  egg  .  .       82 

d.  Preparation  and  employment  of  sterile  blood  serum  .  .       86 

LIQUID  MEDIA  : 

e.  Preparation  of  sterilised  bouillon,  liquid  blood  serum,  urine,  milk, 

vegetable  infusions,  and  artificial  nourishing  liquids     .  .       88 

/  Methods  of  storing  and  employing  liquid  media  ;  Lister's  flasks, 
Aitken's  test-tubes,  Sternberg's  bulbs,  Pasteur's  apparatus, 
Miquel's  bulbs  ;  Drop  cultures  ;  Warm  stages  .  .  91 


CONTENTS.  XV 

CHAPTER  VI. 

PAGE 

EXPERIMENTS   UPON  THE  LIVING  ANIMAL         .  .     107 

a.  Inhalation  of  micro-organisms  .....     107 

b.  Administration  with  food   .  .  .  .  .107 

c.  Cutaneous  and  subcutaneous  inoculation      ....     108 

d.  Special  operations     .......     109 

CHAPTER  VII. 

EXAMINATION  OF  ANIMALS  EXPERIMENTED  UPON, 
AND  THE  METHODS  OF  ISOLATING  MICRO-ORGAN- 
ISMS FROM  THE  LIVING  AND  DEAD  SUBJECT 

a.  Method  of  dissection  and  examination         .  .  .  .in 

b.  Isolation  of  micro-organisms  from  the  living  subject          .  .114 


PART     I  I. 
GENERAL  BIOLOGY  OF  BACTERIA. 

CHAPTER  VIII. 
GENERAL  MORPHOLOGY  AND  PHYSIOLOGY  .  .117 

CHAPTER  IX. 
ANTISEPTICS  AND  DISINFECTANTS  .  .  .  .150 

CHAPTER  X. 
IMMUNITY  162 


PART     III. 

SYSTEMATIC  AND  DESCRIPTIVE,    WITH  SPECIAL 
MICROSCOPICAL  METHODS. 

CHAPTER  XL 
CLASSIFICATION  OF  BACTERIA  .  .  .175 


XVI  CONTENTS. 

CHAPTER  XII. 

PAGE 

SYSTEMATIC  AND  DESCRIPTIVE         .  .  .  .194 

GROUP  I.    COCCACE,E    .  .           .           .           ,           .           .     194 

Genus    I.  Streptococcus  ......     195 

,,       II.  Merismopedia  .             .             ...             .             .     208 

,,     III.  Sarcina     .  .             .            .            .             .     210 

,,      IV.  Micrococcus  .            .             .            .             .            .213 

,,          V.    ASCOCOCCUS  .  .  .  .  .  .       222 

GROUP  II.    BACTERIACE^E  ......    224 

Genus    I.  Bacterium  ......     225 

,,       II.  Spirillum  .  .             .....     249 

,,     III.  Leuconostoc  ......     262 

„      IV.  Bacillus     .  .             .                          .             .             .265 

,,       V.  Vibrio       .......     317 

,,      VI.  Clostridium  .             .             .             .             ^             .318 

GROUP  III.    LEPTOTRICHE^E  .  .  .  .  .  .321 

Genus     I.  Crenothrix  ......     322 

,,       II.  Beggiatoa  ......     324 

,,     III.  Phragmidiothrix  ......     328 

„     IV.  Leptothrix  .  .  .  .  .     329 

GROUP  IV.    CLADOTRICHE/E     ......     330 

Genus     I,  Cladothrix  .  .  .  .  .  .     330 


APPENDICES. 

A.  Yeasts  and  moulds     .......  340 

B.  Flagellated  protozoa  in  the  blood      .....  350 

C.  Examination  of  air  .  .  .  .  .  .361 

D.  Examination  of  soil    .             .             .             .             .             .             .  365 

E.  Examination  of  water              ......  366 

F.  Chronological  bibliography     ......  370 

G.  Table  showing  the  magnifying  power  of  Zeiss'  objectives    .             .  430 


LIST     OF     ILLUSTRATIONS. 

PLATES. 

PLATE 

I.  Bacteria,  Schizomycetes,  or  Fission-fungi      .  .  .  Frontispiece 

II.  Cultivations  on  nutrient  agar-agar. 
Fig.  I.  Bacterium  indicum. 
,,    2.  Bacillus  cyanogenus. 
,,    3.  Bacterium  prodigiosum. 

III.  Cttltivations  in  mitrient  gelatine. 

Fig.  i.  Spirillum  cholerse  asiaticae. 
,,    2.  Bacterium  cholerse  gallinarum. 
„    3.  Streptococcus  cereus  albus. 

IV.  Cultivations  in  nutrient  gelatine. 

Fig.  i.  Micrococcus  tetragonus. 
,,    2.  Bacterium  pneumonise  crouposae. 
,,    3.  Saccharomyces  niger. 

V.   Cultivations  in  nutrient  gelatine. 
Fig.  i.  Bacillus  pyocyaneus. 
,,    2.  Sarcina  lutea. 
,,    3.  Bacillus  anthracis. 

VI.  Plate-cultivation. 

First    attenuation    of    the    Spirillum    Finkleri    after    twenty- four 
hours. 

VII.  Plate-ctiltivation. 

Second    attenuation   of    the    Spirillum    Finkleri    after    thirty-six 
hours. 

VIII.   Cultivations  on  nutrient  agar-agar. 
Fig.  I.  Sarcina  lutea. 
,,    2.  Streptococcus  pyogenes  aureus. 
,,    3.  Bacillus  pyocyaneus. 

IX.  Potato-cultivations. 

Fig.  i.  Bacterium  prodigiosum. 
,,     2.  Penicillium  glaucum. 

b 


XV111  LIST    OF    ILLUSTRATIONS. 

PLATE 

X.  Potato  cultivations. 

Fig.  I.  Sarcina  lutea. 

,,    2.  Saccharomyces  rosaceus. 
XL  Bacillus  tuberculosis. 

Fig.  i.  Pure  cultivation  on  solid  blood  serum  in  a  test-tube. 
,,    2.   Pure  cultivation  on  solid  blood  serum  in  a  glass-capsule. 
,,    3.  The  same  preparation  as  Fig.  2  (  x  80). 
,,     4.  Cover-glass   impression-preparation   of    colonies   (  x  700). 

Ehrlich's  method. 
XII.  Micrococcus  tetragonus  and  Streptococcus  pyogenes  aureus. 

Fig.  i.  From  a  section  of  kidney  of  a  mouse.     Gram's  method. 
,,    2.   From  a  section  of  liver  of  a  rabbit.     Gram's  method. 

XIII.  Cultivations  on  nutrient  agar-agar. 

Fig.  I.  Sarcina  lutea. 
„    2.  Bacterium  indicum. 
„     3.  Saccharomyces  rosaceus. 

XIV.  Cultivations  on  nutrient  agar-agar. 

Fig.  I.  Bacillus  anthracis. 
,,    2.  Bacillus  subtilis. 
,,    3.  Streptococcus  cereus  albus. 
XV.  Potato-cultivations. 

Fig.  I.  Bacillus  anthracis. 

,,    2.  Bacterium  indicum. 
XVI.  Bacillus  anthracis. 

Fig.  i.  From  a  section  of  mucous  membrane  of  the  stomach  of  a 

mouse.     Gram's  method  and  eosin. 
,,    2.  From  a  section  of  kidney  of  a  mouse.     Gram's  method  and 

eosin. 
XVII.  Bacillus  anthracis  and  Micrococcus  tetragonus. 

Fig.  I.  From  a  section  of  liver  of  a  mouse.     Weigert's  and  Orth's 

methods. 
,,    2.  From  a  section  of  lung  of  a  mouse.     Gram's  method  and 

eosin. 
XVIII.  Bacillus  tuberculosis. 

Fig.  I.  From   a   section   of    a   lymphatic   gland    from    a   case   of 

tuberculosis  in  a  foetal  calf.     Ehrlich-Koch  method. 
,,    2.  From  a  section  of  lung  from  a  case  of  artificial  tuberculosis 

in  a  rabbit.     Ehrlich-Koch  method. 
XIX.  Bacillus  tuberculosis. 

Fig.  i.  From  a  section  of  liver  of  a  hen.     Ehrlich-Koch  method. 
„    2.  The  same  preparation.     (Zeiss'  ^  o.i.  Oc.  4.) 


LIST    OF    ILLUSTRATIONS.  XIX 

PLATE 

XX.  Bacillus  tuberculosis  and  Bacilli4s  lepra. 

Fig.  I.  From  a  cover-glass-preparation  of  pus  from  a  tubercular 

cavity  of  human  lung. 

,,    2.  From  a  section  of  kidney  from  a  case  of  leprosy.    Ehrlich's 
method. 

XXI.  Potato-cultivation  of  Bacillus  cyanogenus. 
Fig.  I.  After  three  days'  growth. 
,,    2.  After  nine  days'  growth. 

XXII.  Bacillus  of  septicamia  of  mice. 

Fig.  i.  From  a  section  of  kidney  of  a  mouse.     Gram's  method 

and  eosin. 
,,    2.  The  same  preparation.     (Zeiss'  rV  o.i.  Oc.  4  ) 

XXIII.  Bacillus  lepra. 

Fig.  I.  From  a  section  of  skin  from  a  case  of  leprosy.     Ehrlich's 

method. 
,,    2.  The  same  preparation.     (Zeiss'  TV  o.i.  Oc.  4.) 

XXIV.  Cultivations  on  nutrient  agar-agar. 

Fig.  I.  Bacterium  lineola. 
,,    2.  Micrococcus  rosaceus. 
,,    3.  Streptococcus  pyogenes  citreus. 

XXV.  Bacillus  figurans. 

Fig.  I.  Cover-glass    impression-preparation    from   a   plate-culti- 
vation. 
,,    2.  The  same  preparation.     (Zeiss'  ^  o.i.  Oc.  4.) 

XXVI.  Bacillus  of  swine- erysipelas. 

Fig.  I.  Pure  cultivation  in  nutrient  gelatine. 
,,    2.  Colonies  on  a  plate-cultivation. 

,,    3.  Cover-glass-preparation    of   blood    from    an    inoculated 
pigeon. 

XXVII.  Actinontyces. 

Fig.  i.  From  a  section  of  a  maxillary  tumour  in  a  cow.     Plaut's 

method. 
,,    2.  The  same  preparation.     (Zeiss'  ^  o.i  Oc.  2.) 

XXVIII.  Actinomyces. 

Fig.  i.  From  a  section  of  a  maxillary  tumour  in  a  cow.    Weigert's 

method. 
,,    2.  From  a  section  of  the  lung  of  a  cow.     Weigert's  method. 

XXIX.    Yeast-fungi  or  Saccharomycetes,  and  Mould-fungi  or  Hyphomycetes. 

facing  page  339 


XX  LIST    OF    ILLUSTRATIONS. 


WOOD    ENGRAVINGS. 


FIG.  PAGE 

1.  Koch's  Steam-Steriliser  .  .  .  .  .  19 

2.  Hot-air  Steriliser  .......       20 

3.  Section  of  Hot-air  Steriliser      .  .  .  .  .  .20 

4.  Hot-water  Filtering  Apparatus,  with  Ring  Burner      .  .  .22 

5.  Wire  Cage  for  Test-tubes          .  .  .  .  .  .24 

6.  Platinum  Needles          .  .  .  .  .  .  25 

7.  Damp-chamber  for  Plate-cultivations  .  .  .  .  .26 

8.  Apparatus  employed  for  Plate-cultivations       .  .  .  .26 

9.  Box  for  Glass  Plates     .......       27 

10  and  II.  Glass  Benches  for  Glass  Plates  or  Slides  .  .  -27 

12.  Israel's  Case      ........       28 

13.  Damp-chamber,  for  Potato-cultivations  .  .  .  .28 

14.  Serum  Steriliser  ......  .29 

15.  Serum  Inspissator          .  .  .  .  .  .  -3° 

16.  D'Arsonval's  Incubator  ......       33 

17.  Schlosing's  Membrane  Regulator          .  .  .  .  •       34- 

1 8.  Gas-burner  protected  with  Mica-cylinder         .  .  .  •       35 

19.  Moitessier's  Gas-pressure  Regulator     .  .  .  .  36 

20.  Koch's  Safety  Burner    .  .  .  .  .  .36 

21.  Babes'  Incubator  .  .  .  .  .  -37 

22.  Reichert's  Thermo-Regulator   .  .  .  .  .  -39 

23.  Meyer's  Thermo-Regulator       .  .  .  .  .  .40 

24.  Siphon   Bottle,  with   Flexible   Tube,   Glass   Nozzle,  and  a   Mohr's 

Pinchcock     ........       42 

25.  Dessicator          ........       43 

26.  Method  of  making  a  Folded  Filter       .  .  .  .  -67 

27.  Method  of  Inoculating  a  Test-tube,  containing  sterile  nutrient  jelly    .       70 

28.  Method  of  Inoculating  Test-tubes  in  the  preparation  of  Plate-culti- 

vations         .  .  .  .  .  .  .  -75 

29.  Microscopical   Examination  of  Colonies  on    Plate-cultivations,  and 

Method  of  Isolating  Colonies  by  Inoculation  of  Test-tubes          .       79 

30.  Method  of  dividing  Potatoes     .  .    »  .  .  .84 

31.  Method  of  Forming  a  Simple  Moist  Chamber  .  .  -97 

32.  Simple  Warm  Stage      .......       (,8 


LIST    OF    ILLUSTRATIONS.  XXI 

FIG.  PAGE 

33.  Simple  Warm  Stage  shown  in  Operation         .            .            .  •       99 

34.  Schafer's  Warm  Stage  .             .             .             .             .-           .  .100 

35.  Strieker's  Warm  Stage              .            .             .             .             .  .100 

36.  Section  of  Israel's  Warming  Apparatus  and  Drop-culture  Slide  .     101 

37.  Israel's  Warming  Apparatus     .            .            ..        .         ..  .101 

38.  Israel's  Warming  Apparatus  in  Operation        .             .             .  .102 

39.  Simple  Gas  Chamber                 .'  •           .             .             »            .  .     103 

40.  Gas  Chamber  in  use  with  Apparatus  for  generating  Carbonic  Acid  .     104 

41.  Strieker's  Combined  Gas  Chamber  and  Warm  Stage  .             .  .     105 

42.  Simple  Moist  Chamber  adapted  for  transmission  of  Electricity  .     105 

43.  Apparatus  arranged  for  transmitting  Electricity           .             .  .     106 

44.  Slide  with  Gold-leaf  Electrodes            .             .             .             .  .106 

45.  From  a  Preparation  of  Bacillus  anthrads        .             .            .  .119 

46.  Ascococcus  Billrothii,   x  65  (after  Cohn)          .             .             .  .122 

47.  Streptococcus  and  Sarcinacoccus  from  a  Drop-cultivation,   x   1200  .     123 

48.  Streptococcus  in  the  Blood  of  a  Rabbit,  x  1200          .             .  .     123 

49.  Streptococcus  of  Progressive  Tissue  Necrosis  in  Mice  (after  Koch)  .     123 

50.  Spirochaeta  from  Sewage  Water,   x  1200          .             .             .  .125 

51.  Bacteria  showing  Flagella         .             .             .             .             .  .127 

52.  Bacillus  megaterium      .             .             .             ...             .  .129 

53.  Clostridium  butyricum,  x   1020             .             .             .             .  .130 

54.  A  Thread  of  Bacillus  anthrads  with  Spores  in  a  Drop- cultivation, 

x  1400        .  .  .  .  .  -131 

55.  Leuconostoc  Mesenteroides  ;  Cocci-chains  with  Arthrospores  (after 

Van  Tieghem  and  Cienkowski)      .  .  .  .  .     131 

56.  Spores   of  Bacillus  anthrads,   stained   with   Gentian  Violet,    after 

passing  the  cover-glass  twelve  times  through  the  flame,  x  1200    .     133 

57.  Spore-bearing  Threads  of  Bacillus  anthrads  ^   Double-stained  with 

Fuchsine  and  Methylene  Blue,  x  1200      .  .  .    .         .  133 

58.  Tubercle  Bacilli  in  Sputum,  x  2500  (from  photographs)        .  .  135 

59.  Leprosy  Bacilli  from  a  Section  of  Skin,   x  1200          .  .  .  135 

60.  Glanders  Bacilli  from  a  Section  of  a  Glanders  Nodule,   x    1200  .  135 

61.  Bacterium  of  Chicken-cholera  from  Blood  of  Infected  Hen,  x  1200   .  136 

62.  Bacterium  of  Chicken-cholera  from  Muscle  Juice  of  an  Infected  Hen, 

x  2500  (from  a  photograph)          .             .             .  .  .136 

63.  Comma  Bacilli  in  Sewage  Water  stained  with  Gentian  Violet,  x  1200     136 

64.  Vibrios  in  Water  contaminated  with  Sewage,  x  1200.  .  .     137 

65.  Spirillum  undula,  x  1200         .             .             .             .  .  .     137 

66.  Cladothrix  dichotovia    .             .             .             .             .  .  .184 

67.  Bacterium  pneumonias  crouposce^  x  1500  (after  Zopf)   .  .  .     189 

68.  Emmerich's  Bacterium,  x  700  (after  Emmerich)          .  .  .     190 


XX11  LIST    OF    ILLUSTRATIONS. 

FIG.  PAGE 

69.  Colonies  on  Nutrient  Gelatine,  x  60    ,  .  .  .  .190 

70.  Colonies  on  Nutrient  Agar-agar,  x  60  .  .  .  .191 

71.  Colony  of  Bacillus  anthracis,  x  60       .  .  .  .  .191 

72.  Bacterium  of  Rabbit  Septicaemia  .....     192 

73.  Streptococcus  of  Progressive  Tissue  Necrosis  in  Mice  .  .     206 

74.  Micrococcus  of  Pyaemia  in  Rabbits  ;  Vessel  from  the  Cortex  of  the 

Kidney,  x  700        .  .  .  .  .  .  .217 

75.  Ascococcus  Billrothii  (after  Cohn)          .....     222 

76.  Bacterium  Pneumoniae  Crouposae,  from  Pleural  Cavity  of  a  Mouse. 

x  1500  (after  Zopf)  ......     226 

77.  Bacterium  Neapolitanum,  x  700  (after  Emmerich)     .  .  .     229 

78.  Bacteria  of  Rhinoscleroma,  x  1400  (after  Cornil)       .  .  .229 

79.  Bacterium  of  Chicken  Cholera  ;  Blood  of  Inoculated  Hen,  x  1200    .     233 

80.  Bacterium  of  Chicken  Cholera,  from  Muscle  Juice  of  Inoculated  Hen, 

x  2500       .  .  .  .  .  .  .  -233 

81.  Bacterium  of  Rabbit  Septicaemia  ;  Blood  of  Sparrow,  x  700  (after 

Koch)          ......  -235 

82.  Bacterium  Indicum  ;  Colonies  on  Nutrient  Agar-agar,  x  60   .  .     242 

83.  Bacterium  Zopfii ;  Successive  Changes  in  the  same  Thread,  x  740     .     245 

84.  Cover-glass  Preparation  of  the  Edge  of  a  Drop  of  Meat  Infusion, 

x  600  (after  Koch)  .  .  .  .  .  -251 

85.  Colonies  of  Comma  Bacilli  on  Nutrient  Gelatine,  natural  size  (after 

Koch)          .  .  .  .  .  .  .  .251 

86.  Colonies  of  Koch's  Comma  Bacilli,  x  60  .  .  .     252 

87.  Cover-glass  Preparation  from  the  Contents  of  a  Cholera  Intestine, 

x  600  (after  Koch)  ......     252 

88.  Cover-glass  Preparation  of  Cholera  Dejecta  on  Damp   Linen  (two 

days  old),  x  600  (after  Koch)         .....     252 

89.  Section  of  the  Mucous  Membrane  of  a    Cholera  Intestine,  x  600 

(after  Koch)  .  .  .  .  .  .  .253 

90.  Pure  Cultivation  of  Finkler's  Bacillus,  twenty -four  hours  old .  .     254 

91.  „  „  ,,  two  days  old     .  .     254 

92.  ,,  Koch's  Cholera  Bacillus,  twenty-four  hours  old    .     254 

93.  ,,  ,,  ,,.  two  days  old      .  .     254 

94.  Comma-shaped    Organisms    with    other    Bacteria    in   Sewage-con- 

taminated water,  x  1200     .  .  .  .  .  .256 

95.  Pure  Cultivation  of  Spirillum  Finkleri  in  twenty-four  hours    .  *     258 

96.  ,,  ,,  ,,  thirty-six  hours       .  .     258 

97.  Spirillum  sputigenum,  x  1 2OO  ......     259 

98.  Spirillum  tyrogemtm,  x  I2OO     ......     260 

99..  Spirillum  plicatile  (Marsh  Spirochcete),,  x  1200  .  »  .261 


LIST    OF    ILLUSTRATIONS.  XXlll 

FIG.  PAGE 

100.  Spirillum  undula,  x  1500        ......     262 

101.  Leuconosloc  mesenteroides         .  .  .  .  .  .     263 

1 02.  Leprosy  Bacilli  from  a  Section  of  Skin,  x  1200  .  .  .     266 

103.  Bacillus  typhostts  from  a  Potato-cultivation,  x  1500    .  .  .     269 

104.  Bacillus  tuberculosis  from  Tubercular  Sputum,  stained  by  Ehrlich's- 

method,  x  2500       .  ......  272 

105  and  106.  Bacillus  anthracis,  x  1200     .....  282 

107.  Pure  Cultivation  of  the  Bacillus  anthracis  in  Nutrient  Gelatine         .  284 

108.  Colonies  in  a  Plate-cultivation,  x  70    .  .  ..  .  .  285 

109.  Cover-glass  impression-preparation,  x  70         .             .             .             .  286 
no.  Spores  of  Bacillus  anthracis  unstained,  x  1500            .             .             .  290 

111.  Spores  of  Bacillus  anthracis,  x  1200   .  .  .  .  .291 

112.  From  a  double-stained  preparation  of  Bacillus  anthracis,  x  r20O       .     291 

113.  Bacillus  Mallei,  x  1 200  ......     292 

1 14  and  1 15.   Pure  Cultivations  of  the  Bacillus  of  Septicaemia  of  Mice      .     298 

1 16.  From  a  preparation  of  Bronchial  Mucus  of  a  Pig  (after  Klein)          .     299 

117.  Blood  of  Fresh  Spleen  of  a  Mouse,  after  inoculation  with  Swine 

Fever  (after  Klein) .             .  \                     .             .             .  .     300 

118.  Bacilli  from  an  Artificial  Culture  with  Spores  (after  Klein)  .  .     300 

119.  Bacillus  cyanogenus,  x  650  (after  Neelsen)      ....     305 

1 20.  Bacillus  megaterium  (after  De  Bary)  .             .             .            «  .310 

121.  Pure  Cultivation  of  Bacillus  jigurans  in  Nutrient  Agar-agar  .     311 

122.  Bacillus  saprogenes^  No.  I  (after  Rosenbach)              .             .  .   .3^4 

123.  Vibrio  rugula,  x  1020  (after  Prazmowski)        .             .             ..  .     317 

124.  Clostridium  butyricum  (after  Prazmowski)      .             ..            .  -319 

125.  Crenothrix  Kilhniana  (after  Zopf)      .  323 

126.  Beggiatoa  alba  (after  Zopf)      .  325 

127.  Several  Phase-forms  of  Beggiatoa  Roseopersidna  (after  Warming),    .     326 

128.  Cladothrix  dichotoma  (after  Zopf)        .             .             .             .  .     331 

1 29.  Parasites  in  the  Blood  of  Rats  (after  Lewis)    .             .             .  .     35 1 

130.  Hiematomonas  cobitis  (after  Mitrophanow)      .             .             .  -353 

131.  Organisms  in  the  Blood  of  the  Carp  (after  Mitrophanow)      .  .     354 

132.  "  Surra  "  Parasites  occur  ring  singly  and  fused,  x  1200           .  .     356 

133.  A  Monad  in  Rat's  Blood,  x  3000         .             .             .             .  -357 
134  and  135.  Monads  in  Rat's  Blood,  x  1200          .                          .  .     358 

136.  Hesse's  Apparatus       .......     363 

137.  Apparatus  for  estimating  the  number  of  Colonies  on  a  Plate-culti- 

vation         .  .  .  .  .  .  .  .     3^8 


DESCRIPTION    OF    PLATE    I. 


FIG. 

1.  Cocci  singly  and  varying  in  size. 

2.  Cocci  in  chains  or  rosaries  (streptococcus). 

3.  Cocci  in  a  mass  or  swarm  (zooglcea). 

4.  Cocci  in  pairs  (diplococcus). 

5.  Cocci  encapsuled  {Bacterium  pneumonia  crouposa], 

6.  Cocci  in  groups  of  four  (merismopedia). 

7.  Cocci  in  packets  (sarcina). 

8.  Bacterium  termo. 

9.  Bacterium  termo,  x  4000  (after  Dallinger  and  Drysdale). 

10.  Bacterium  lineola. 

11.  Bacillus  sublilis. 

12.  Bacillus  tuberculosis. 

13.  Bacillus  lepra. 

14.  Bacillus  malaria  (after  Klebs). 

15.  Bacillus  typhosus  (after  Eberth). 

1 6.  Spirillum  undula  (after  Cohn). 

17.  Spirillum  volutans  (after  Cohn). 

1 8.  Spirillum  cholera  Asiatics:  from  an  artificial  cultivation. 

19.  Spirillum  Obermeieri  (after  Koch). 

20.  Spirochate  plicatilis  (after  Fliigge). 

21.  Vibrio  rugula  (after  Prazmowski). 

22.  Cladothrix  Fcerstcri  (after  Cohn). 

23.  Cladothrix  dichotoma  (after  Cohn). 

24.  Monas  Okenii  (after  Cohn). 

25.  Monas  Warmingii  (after  Cohn). 

26.  Rhabdomonas  rosea  (after  Cohn). 

27.  From  a  cover-glass-preparation  of  blood  from  the  spleen  of  a  mouse  which 

had  died  of  anthrax ;  stained  with  fuchsine  (Zeiss'  ^  o.i.  Oc.  4). 

28.  From  a  drop-cultivation  of  Bacillus  anthracis  (Zeiss'  TV  o.i.  Oc.  4). 

29.  From   a   cover-glass    impression-preparation    of    a   potato- cultivation    of 

Bacillus  anthracis  (Zeiss'  ^  o.i.  Oc.  4). 

30.  From  a  preparation  of  Bacillus  anthracis,  cultivated  in  nutrient  gelatine 

(torula-form). 

31.  Involution-form  of  Crenothrix  (after  Zopf). 

32.  Involution-forms  of  Vibrio  serpens  (after  Warming). 

33.  Involution-forms  of  Vibrio  rugula  (after  Warming). 

34.  Involution-forms  of  Clostridium  Polymyxa  (after  Prazmowski). 

35.  Involution-forms  of   the    Spirillum  cholera  Asiatica,    from  an    artificial 

cultivation. 

36.  Involution-forms  of  Bacterium  aceti  (after  Zopf  and  Hansen). 

37.  Spirulina-form  of  Beggiatoa  alba  (after  Zopf). 

38.  Various  thread-forms  of  Bacterium  merismopedioides  (after  Zopf). 

39.  False-branching  of  Cladothrix  (after  Zopf). 


DESCRIPTION   OF   PLATES. 


PLATE    I. 

FIGS,  i  to  39. 

Bacteria,  Schizomycetes,  or  Fission  fungi, 

(Facing  Title-fiage.) 
For  description,  see  p.  xxiv. 


PLATE  II. 

FIG.   i. — Bacterium  indicum. 

Micrococcus  indicus. 

Bacillus  indicus. 

Tube  inoculated  from  a  nutrient  agar-agar  plate- 
cultivation.  By  plate-cultivation,  or  by  successive 
cultivation  on  potatoes,  a  pure  cultivation  can  be 
obtained.  The  growth  has  then  the  colour  of  red 
sealing-wax,  and  a  peculiar  crinkled  appearance. 
After  some  days  the  growth  loses  its  bright 
colour,  and  becomes  purplish  like  an  old  cultiva- 
tion of  Bacterium  prodigiosum. 

FIG.  2. — Bacillus  cyanogenus. 

Bacterium  syncyanum. 
Bacillus  of  blue  milk. 

Tube  inoculated  from  a  potato-cultivation.  The  bacillus 
forms  a  whitish  layer,  and  colours  the  nutrient 
agar-agar  a  smoky  brown. 

FIG.  3. — Bacterium  prodigiosum. 

Monas  prodigiosa. 

Micrococcus  prodigiosus. 

Bacillus  prodigiosus. 

Blood  rain. 

Tube  inoculated  from  a  potato  cultivation.  The  bacterium 
grows  very  rapidly,  forming  a  blood-red  growth, 
which  gradually  acquires  a  purplish  colour. 


CULTIVATIONS  ON  miTKLENT  AGAR-A.GAR 

Fig  1.  Micrococcus  indicus. 
Fig  2.J3sucillus  cyanogenus. 
Fig  3 .  Micro  coccus  prodigiosus. 

i 

••  ~f  ?•••->•(•. 


PLATE  III. 

FIG.  i. — Spirillum  cholerae  asiaticae. 

Comma-bacillus  of  Koch. 

Tube  inoculated  from  a  plate-cultivation.  The  growth 
in  this  case  was  very  striking.  The  funnel-shaped 
area  of  liquefaction,  enclosing  an  air-bubble,  and 
the  white  thread  along  the  needle  track,  are  in 
marked  contrast  to  the  appearances,  under  similar 
conditions,  of  the  comma-bacillus  in  Cholera 
nostras.  (Fig.  96.) 

FIG.  2. — Bacterium  cholerae  gallinarum. 

Micrococcus  cholerce  gallinarum. 
Microbe  dn  cholera  des  poules. 

Tube  inoculated  from  the  blood  of  a  hen  which  had  died 
of  so-called  chicken -cholera.  After  several  days  the 
growth  forms  a  very  delicate,  finely  beaded  thread. 

FIG.  3. — Streptococcus  cereus  albus. 

Staphylococcus  cereus  albus. 

Tube  inoculated  from  the  pus  of  a  subcutaneous  abscess 
in  a  rabbit.  The  growth  assumed  a  nodular  appear- 
ance along  the  needle  track. 


Rgl 


3. 


CULTIVATIONS    IN  NUTRIENT  GELATINE 

Fig  1.  Spirillum  cholerae  asialacse. 
Fig  2.Micrococcus  cholerae  gallmarum . 
Pig  3.  Stapl^lococcas  cereus  albus. 


PLATE  IV. 

FIG.  i. — Micrococcus  tetragonus. 

Tube  inoculated  from  a  plate-cultivation  of  bacteria  in 
sputum.  The  cultivation  consisted  of  a  milk-white 
growth  heaped  up  on  the  surface  of  the  gelatine  and 
growing  freely  along  the  upper  part  of  the  needle 
track. 

FIG.  2. — Bacterium  pneumoniae  crouposae. 

Micrococcus  pneumonia  crouposce. 
Friedldnders  pneumo-  coccus. 

Tube  inoculated  from  pneumonic  exudation.  The  growth, 
in  nutrient  gelatine,  in  the  form  of  a  round-headed 
nail  is  not  by  itself  distinctive.  • 

FIG.  3. — Saccharomyces  niger. 

Black  torula. 

Tube  inoculated  from  an  old  contaminated  nutrient 
gelatine  cultivation.  The  growth,  isolated  and  re- 
inoculated,  formed  a  black  crust  on  the  surface  of 
the  gelatine.  In  some  of  the  tubes  little  separated 
centres  of  growth  occurred  in  the  upper  part  of 
the  track  of  the  needle. 


Figl. 


Fig 


2. 


CULTIVATIONS    IN  NUTRIENT  GELATINE. 

Fid  1.  Micro  coccus  tetragonus. 

fid  2.  Bacterium  pneumoniae  crouposae. 

Fid  3.  Sacoharamyees  niger. 


PLATE  V. 

FIG.  i. — Bacillus  pyocyaneus. 

Bacterium  ceruginosum. 
Bacillus  fluorescens. 

Tube  inoculated  from  pus.  The  gelatine  was  liquefied 
and  appeared  green  by  transmitted  and  orange  by 
reflected  light. 

FIG.  2. — Sarcina  lutea. 

Tube  inoculated  from  a  colony  which  occurred  on  potato 
exposed  to  the  air.  The  gelatine  was  partially 
liquefied,  and  a  canary-yellow  growth  had  subsided 
to  the  bottom  of  the  liquefied  layer. 

FIG.  3. — Bacillus  anthracis. 

Tube  inoculated  from  the  blood  of  a  mouse  which  had 
died  of  anthrax.  The  typical  growth  which  occurs 
in  a  few  days  is  shown  in  Fig.  107.  In  this  figure 
the  appearance  after  three  weeks  is  represented. 
The  gelatine  was  completely  liquefied,  and  a  floc- 
culent  mass  had  subsided  to  the  bottom  of  the 
tube. 


Fif5  2. 


Fig  3. 


CULTIVATIONS  IN  NUTRIENT   GELATINE 

Pig  1.  Bacterium  aeru^mosum. 
Tig  2.  Sarcina  lutea. 
B.£  3.  BaciTlus  sunthracis . 


PLATE  VI. 
Spirillum  Finkleri. 

Comma-bacillus  of  Finkler  and  Prior. 

This  figure  represents  the  appearance  of  a  plate-cultiva- 
tion of  the  comma-bacillus  from  Cholera  nostras,  when 
examined  over  a  slab  of  blackened  plate-glass.  The 
colonies  differ  very  markedly  from  the  colonies  of 
Koch's  comma-bacilli  (see  Fig.  85).  The  drawing 
was  made  from  a  typical  result  of  thinning  out  or 
attenuating*  the  colonies  by  the  process  of  plate- 
cultivation.  At  this  stage  they  were  completely 
isolated  one  from  the  other  ;  but  later  they  became 
confluent  and  produced  complete  liquefaction  of  the 
gelatine. 

*  The  term  "  attenuation"  is  applied  also  to  a  virus,  in  the  sense 
of  weakening  or  modifying  its  effect.  To  avoid  confusion  the  term 
mitigation  might  be  employed  exclusively  to  express  this,  and 
attenuation  used  only  in  the  sense  indicated  above. 


PLATE  -  CULT  IV  AT  I  ON. 

Ftrst  attenuation  of  the  Spirillum  FtnJderii,  after  twenty- four-  hours. 


LondoixRiblishedbv  HJLLevri3.136.Goww  Street. 


Vincent  Brvolej.Bty  SeSon.Utk. 


PLATE  VII. 

Spirillum  Finkleri. 

Comma-bacillus  of  Finkler  and  Prior. 

This  figure  represents  the  result  obtained  by  a  still 
further  thinning  out  of  the  organisms  than  in  the 
preceding  case.  The  attenuation  had  been  so  far 
carried  out  that  several  of  the  colonies  remained 
completely  isolated  for  days. 


PLATE      CULTIVATION. 

Second  attenuation  of  the  Spirillum  Finklerii .  a/fter  thirty -sue  fiourn. 


LondoixPatlislied  by  HJLLevvis,l36.Gower  Street . 


Knceni  Bnolcs.Ilay  itSon.,LMi 


PLATE  VIII. 

FIG.  i. — Sarcina  lutea. 

Tube  of  nutrient  agar-agar  inoculated  from  a  plate- 
cultivation.  The  canary-yellow  colour  forms  a 
strong  contrast  to  the  colour  of  the  growth  in  the 
adjacent  tube. 

FIG.  2. — Streptococcus  pyogenes  aureus. 

Staphylococcus  pyogenes  aureus. 
Micrococcus  pyogenes  aureus. 

Tube  inoculated  from  an  abscess  in  a  rabbit 

FIG.  3. — Bacillus  pyocyaneus. 

Bacillus  fluorescent. 

Bacterium  aeruginosum. 

Tube  inoculated  from  a  colony  on  a  plate-cultivation. 
The  growth  formed  a  whitish,  transparent  layer 
composed  of  slender  bacilli.  The  pigment  diffused 
itself  throughout  the  nutrient  jelly.  The  growth 
appears  green  by  transmitted  light  owing  to  the 
colour  of  the  medium  behind  it.  The  bacillus  is 
now  regarded  as  identical  with  the  bacillus  of 
green-blue  pus. 


>LATF.  8. 


CULTIVATIONS  ON  NUTRIENT  AGAR-AGAR. 

Pif5  1 .  Sareina  lutea . 

Fig  2.  Micrococcus  pyogen.es  aureus. 

Fid  3 .  Bacillus  fhiorescens . 


Vincent  Broo'ks.Day  l<Son.,U'th. 


PLATE  IX. 

FIG.  i. — Bacterium  prodigiosum, 

Monas  prodigiosa. 

Micrococcus  prodigiosus. 

Bacillus  prodigiosus. 

Blood  rain. 

Growth  on  potato  after  three  days. 


FIG.  2. — Penicillium  glaucum. 

A  potato  which  had  been  freely  exposed  to  the  air  was 
covered  in  three  weeks  by  a  growth  of  Penicillium 
glaucum.  The  surface  of  the  growth  is  studded 
with  dew-like  drops  of  moisture. 


-LA1E  9. 


^  1.  Micro co  ecus  prodigiosus. 
Second  attenuation  after  three  days 


Fig  2.  Peiu-cillium 

after  three  weeks  grovrih/. 


POTATO     CULTIVATIONS. 


London.PuhEs'hed  bv  H  .K  Lews  .  1  3  6  .  Gower  Street  . 


PLATE  X. 

FIG.  i. — Sarcina  lutea. 

Growth  on  sterilised  potato  five  days  after  inoculation 
from  a  tube-cultivation.  Potatoes,  especially  old 
ones,  have  sometimes  a  tendency  to  become  dis- 
coloured, and  the  brown  appearance  in  this  figure  has 
nothing  to  do  with  the  growth  of  the  organisms. 

FIG.  2. — Saccharomyces  rosaceus. 

Pink  torula. 

Growth  on  sterilised  potato  which  had  been  inoculated 
from  a  colony  contaminating  a  plate-cultivation. 
This  yeast  develops  a  coral-pink  colour,  but  does 
not  grow  so  luxuriantly  as  the  chromogenic  bacteria. 


LATE  10. 


Fi^l.  Sarcirxa-lutea 
Growth,  five  days  a/tor  vnoculcuUorv. 


Tig  2.    Saccharomyees  rosaceus. 
Growth,  two  days  tftcr  vnocula,li,on. 

POTATO    CULTrVATIONS. 


LondoixRibHskedbv  H.K.Lewis ,136. Gower  Street. 


PLATE  XI. 

Bacillus  tuberculosis. 

FIG.  i. 

Pure  cultivation  of  the  tubercle-bacillus  on  blood-serum 
solidified  obliquely. 

FiG.   2. 

Pure  cultivation  on  solid  blood-serum  in  a  glass  cap- 
sule. 

FiG.   3. 

The   same  as  Fig.   2,  examined  under  a  low  power  of 
the  microscope,      x    80. 

FiG.  4. 

Impression-preparation  showing  the  peculiar  serpentine 

growth  of  the  colonies  on  blood-serum.      x   700. 
(After  Koch,  Mittheil.  a.  d.  Kaiser  L  Gesundh. 


PLATE   11. 


Fifl.4. 
BACILLUS       TUBERCULOSIS. 

Fig.  1 .  Pu/re--&dtiva>twn<  on  solid  blood  s&rwm  in,  a  test-tube. 

Fig.  Z.  twe-cuttivation  on  solid  Hood  serum-  in,  a,  glass -capsule. 

Fig.  3 .  The  same-  preparation  as  fig.  2  fjfSOj. 

Ficf.4.    Cover -glass  impression -prcparalior.  ofwlonies  X  7OO  (Ehrliclis  method). 

Hncmtffroots,  Day  t.  Son, 

foauufoitsemtt.      Lonaor,RibJia  tied  bv  H  -  K.  L^/ns  ,136,  Cover  Street . 


PLATE  XII. 

FIG.  i. — Micrococcus  tetragonus. 

From  a  section  of  a  kidney  of  a  mouse  which  had  died 
in  eight  days,  after  inoculation  subcutaneously  with 
a  pure  cultivation.  Encapsuled  tetrads,  isolated  and 
in  masses,  were  found  in  the  kidneys,  lungs,  and 
other  organs.  Stained  with  Gram's  method  (gentian 
violet)  without  a  contrast  stain,  x  1,500. 

FIG.  2. — Streptococcus  pyogenes  aureus. 

Micrococcus  pyogenes  aureus. 
Staphylococcus  pyogenes  aureus. 

From  a  section  of  the  liver  of  a  rabbit.  A  small  vessel 
is  shown  plugged  with  cocci.  From  small  abscesses 
in  the  liver,  cultivations  were  obtained  of  the 
characteristic  yellow  coccus  of  pus.  Stained  with 
Gram's  method  (gentian  violet)  without  a  contrast 
stain.  x  1,500. 


PLATE    12 


•:- 


MICROCOCCUS      TETRAGOTSTUS 

Fiy.  I.    From.  a.  scctipn.  of  J&dnei/  of 'a  mousse. 
G-ram's   method,   Zeiss '  Is"  o.  v.    Oc.  4-. 


MICROCOCCUS    PYOGENES     AUREUS. 
Fig.  Z.  from  a,  section  of  Liver  of  a,  ra&bit 
Grain 's    -method.    X&iss '  Is.  o.  i.    Oc.   4 . 


Edgar  CrceJcshank  ftc.itpintt. 


.K.Leu'i3,lS6,Gower  Street. 


PLATE  XIII. 

FIG.  i. — Sarcina  lutea. 

In  this  tube  and  the  two  adjacent  ones,  the  inoculations 
were  made  by  thrusting  the  needle  into  the  nutrient 
agar-agar.  In  all  three  cases  the  growth  on  the 
surface,  freely  exposed  to  air,  developed  a  charac- 
teristic pigment,  while  the  growth  in  the  track  of 
the  needle  was  scanty  and  colourless. 

FIG.  2. — Bacterium  indicum. 

Micrococcus  indicus. 
Bacillus  indicus. 

FIG.  3. — Saccharomyces  rosaceus. 

Pink  torula. 


Fi£  o 


CULTIVATIONS    IN  NUTRIENT  AGAR  AGAR 

Fig  1.  Sarcma  lulea. 

Fig  2.  li/Ecro  coccus  indicus. 

P'.f  3.  Syc':hju-orayces  rosaceus. 


• 


PLATE  XIV. 

FIG.  i. — Bacillus  anthracis. 

Bacteridie  dn  char b on. 

Bacillus  of  splenic  fever,  woolsorter's  disease,  or 
malignant  pustule. 

Tube  of  nutrient  agar-agar  inoculated  with  the  blood  of  a 
sheep  which  had  died  of  anthrax.  White  flocculent 
patches  developed,  which  were  entirely  composed  of 
threads  and  spores  of  the  bacilli. 

FIG.  2. — Bacillus  subtilis. 

Tube  inoculated  with  bacilli,  isolated  by  plate-cultivation, 
from  dust  The  bacilli  appeared  to  be  identical  with 
the  hay  bacillus,  but  in  this  case  formed  a  peculiar 
crinkled  layer  along  the  track  of  the  needle. 

FIG.  3. — Streptococcus  cereus  albus. 

Staphylococcus  cereus  albus. 

Tube  inoculated  from  the  discharge  of  a  subcutaneous 
abscess  in  a  rabbit. 


PLATE  1A. 


CULTIVATIONS  ON  NUTRIENT  AGAR-AGAR 

Fi«5  1.  Bacillus   anthr-ELCis . 

Fig  ?..  Bacillus  subtilis  . 

Fid  3.  Staphylococous  cereus  sdbus. 


Knaenl  Bnoks.Day  k 


,•  or 


PLATE  XV. 

FIG.  i. — Bacillus  anthracis. 

Bacteridie  du  charbon. 

Bacillus  of  splenic  fever,  woolsorters  disease  or 
malignant  pustule. 

The  bacillus  of  anthrax  grows  very  rapidly  on  sterilised 
potato,  especially  when  placed  in  the  incubator  at 
the  temperature  of  the  blood.  The  growth  forms  a 
creamy-yellow  layer,  with  copious  spore  formation. 

FIG.  2. — Bacterium  indicum. 

Micrococcus  indicus. 
Bacillus  indicus. 

Sterilised  potato  inoculated  with  a  pure  growth  obtained 
by  successive  cultivations.  Unless  the  growth  is 
quite  free  from  the  presence  of  other  bacteria,  the 
brilliant  red  colour  is  not  obtained. 


PLATE  15. 


Fig  1.  Bacillus  artfhracis. 

Growth/  at  XJ°C  three  days  after  moanlcAum 


Fig  2.Mi.crococctLS  in.cb.cus. 

Growth/  three  days    after 


POTATO      CULTIVATIONS. 


Lonaon.RibKshedbv  H..K.Levria.l36.G<ww  Street. 


PLATE  XVI. 
Bacillus  anthracis. 

Bactcridie  du  charbon. 

Bacillus  of  splenic  fever,  woolsoi  ters  disease,  or 
malignant  pustule. 

FIG.  i. 

From  a  section  of  the  mucous  membrane  of  the 
stomach  of  a  mouse.  The  glandular  capillaries 
are  mapped  out  by  the  bacilli.  Stained  by  the 
method  of  Gram  (gentian  violet),  and  eosin. 
x  500. 

FIG.  2. 

From  a  section  of  a  kidney  of  a  mouse.  Under  a  low 
power  the  preparation  has  exactly  the  appearance 
of  an  injected  specimen.  Under  higher  amplifica- 
tion, the  bacilli  are  seen  to  have  threaded  their 
way  along  the  capillaries  between  the  tubules, 
and  to  have  collected  in  masses  in  the  glomeruli. 
Stained  with  Gram's  method  (gentian  violet),  and 
eosin.  x  500. 


PLATE  16. 


J 


A\  //      /-* 

iii  Y     i      r    > 
IM      /     / 


f  y  )  ;  ;  /  jj>       i 

\      V     I/'/'    A\        '•  I     I 

\  ^  fe   '  I  ,  'K  / 
»/ 


/    /row  ar  section  of  mucous    membrane  oftfic  stoinackofa  mouse. 
Grams   method  and  Basin.  Zeiss '  ii.  o.  i.  Oc.2. 


BACILLUS     ANTHRACIS. 

fig.  £.  From    a    section  cfKictneij  of-  a  mouse. 
Gram's  mathcd  and  Sosin.    Xeiss'  ik.  o.  i.  Oc.  Z. 


^yvis  ,136.  Ga.ver  S  treet . 


PLATE  XVII. 

FIG.  i. — Bacillus  anthracis. 

Bacteridie  du  charbon. 

Bacillus  of  splenic  fever,  woolsorter's  disease,  or 
malignant  pusttilc. 

From  a  section  of  the  liver  of  a  mouse  which  had  died 
after  inoculation  with  a  pure  cultivation  of  the 
bacillus.  The  bacilli  are  seen  to  have  threaded 
their  way  between  the  liver-cells.  The  prepara- 
tion is  triple-stained  by  combining  the  methods 
of  Weigert  and  Orth.  x  500. 

FIG.  2. — Bacillus  anthracis   and  Micrococcus 
tetragonus. 

From  a  section  of  a  lung  of  a  mouse  which  had  been 
inoculated  with  anthrax  three  days  after  inocula- 
tion with  Micrococcus  telragomts.  A  double  or 
mixed  infection  resulted.  Anthrax  bacilli 
occurred  in  vast  numbers,  completely  filling  the 
small  vessels  and  capillaries,  and  in  addition  there 
were  great  numbers  of  the  characteristic  tetrads. 
Stained  with  Gram's  method  (gentian  violet),  and 
eosin.  x  500. 


PLATE  17. 


BACILLUS          ANTHRACIS. 

Fig.  1.  frvrn  a  section  of  Liver  of  a  TTwuse.    Wiigerts  anctOrttls  methods. 
(  (jeatiaji-violet  atid piero-Uthiuni-  carmine.)  Zciss '  f%.  o.  ^.  Oc.  Z. 


BACILLUS   ANTHRACIS  AND  MICROCOCCUS    TETRA&OMTS. 

Fig.  2.  From  a  section  ofLttng  of  a,  mouse . 
Groom's  method  andJZosin.    Zeiss' f%  o.i.  Oc.Z. 


Lonaon.Rjbfehedbv  H.K.Lewir  .I. 


Knccntffreoiks,J?ajf  £  Son.  Lilh. 


PLATE  XVIII. 
Bacillus  tuberculosis. 


FIG.  i. 


+ 


From  a  section  of  a  lymphatic  gland  of  a  foetal  calf. 
The  preparation  was  stained  by  the  Ehrlich- 
Koch  method  (methyl  violet  and  bismarck  brown), 
and  eosin.  The  giant  cell  takes  the  eosin  stain,  the 
nuclei  are  stained  brown,  and  the  bacilli  blue. 
In  the  interior  of  the  giant  cell  are  numerous 
coloured  grains,  the  significance  of  which  is  not 
known,  and  a  number  of  tubercle  bacilli, 
x  1,500. 

For  the  material  from  which  this  preparation  was  made 
the  author  is  indebted  to  Professor  Johne,  by 
whom  an  account  of  this  case  was  published, 
"  Ein  Zweifelloser  Fall  von  Congenitaler  Tuber- 
kulose,"  Fortsch.  d.  Med.,  I  885,  No.  7,  p.  198. 


FIG.  2. 

From  a  section  of  a  lung  of  a  rabbit  after  inoculation 
with  tubercular  sputum.  Caseous  areas  are  seen, 
and  masses  of  bacilli  showing  distinct  beading. 
Stained  by  the  Ehrlich-Koch  method  (methyl 
violet)  without  a  contrast  stain.  x  1,500. 


PLATE   18. 


Fig.  1.  From  «,  section  of  a  lymphatic  glanci  from* 

tuberculosis  in  a  foetal  calf.    JZhrlick^J&c 
(methyl-violet  and  bismarcklrownJarulFosin'.  Z 


cas&  of 
oci. 
o.i.  Oc.4-. 


Jfc 

IP 


BACILLUS       TUBERCULOSIS. 
Fig.  Z.  From  a  section  ofZu/ng  frortvacas^-ofcurtificietl  tui&rulosis  in  a  rabbit. 
met/u>d.(  methyl -violet.)  SCeiss'w  o.v.  Oc.  4-, 


Edgar  Crochhovrd.  fic.etpvna. .  Ttnuntffniots,2>ayJI.Son 

Lonaon.Ribli«liedliv  H.KLewi3,136,GoivBr  Street . 


PLATE  XIX. 

Bacillus  tuberculosis. 

FIG.  i. 

From  a  section  of  the  liver  of  a  tubercular  hen. 
With  a  moderate  power  the  areas  of  caseation 
and  the  topographical  distribution  of  the  bacilli 
can  be  studied.  Stained  with  the  Ehrlich-Koch 
method  (methyl  violet  and  bismarck  brown),  x 
400. 

FIG.  2. 

From  the  same  preparation  with  high  amplification, 
showing  that  the  parts  stained  blue  consist 
entirely  of  bacilli,  x  1,500. 


PLATE   19. 


.  fig.l.    from  a  section  of  Liver  ofa>  hen,. 
Ehrlich-KocJv  method  (  methyl -violet  and  lismwck-lroivn,)  %eu>s' DD.  Oc.  4-. 


BACILLUS       TUBERCULOSIS. 

g.  2.     The  same  jtreparcutiori .  Zei$s'  j&.  Oe.  4-. 


HrtuntSrooks,  £>ay  t  Son.. 

Ionaon.RjbK3hedbvH.K.Lewi8,136.Gou»er  Street. 


PLATE  XX. 

FIG.  i. — Bacillus  tuberculosis. 

From    a    cover-glass    preparation    of   tubercular    pu< 
Stained    with    Ehrlich's    method     (fuchsine    and 
methylene  blue),      x    1,500. 

FIG.  2. — Bacillus  leprae. 

From   a  section  of  a  kidney  from  a  case  of  leprosy 
Stained    with    Ehrlich's    method     (fuchsine    and 
methylene  blue).      In   the   centre   of  the  field  is 
a  glomerulus    with    a    collection    of  the   leprosy 
bacilli,     x  400. 


PLATE  20. 


BACILLUS      TUBERCULOSIS. 

xty.  /.    From  CL  cover-qlass  prepara-tivn  of  pus  from  a,  tuvcrcujldr  fui'it-u  of  the 
human,  hwiq.    JEJirlvck's  method./ fuchsirK.  and  metkylene.  blue).  Zeiss'J^.  o.i.  Oc.  4. 


\ 


BACILLUS 

Fig.  2.  Front  a  section  of  Kidney   from-  a  case   oflepn>sy. 
Ekrltch's  method. ( fit chsine  and  metfylene  IrluaJ.  Z&iss'  DD.  Oc.  4- 


jar  Craclahank  be.  etpinx,.  flnanbBro^,Day  &  Son, 

Lonaon.Putiiiliedbv  HJLLewi8,136,Gwwr  Street . 


PLATE  XXI. 


Bacillus  cyanogenus. 

Bacterium  syncyanum. 
Bacillus  of  blue  milk. 


FIGS,  i  and  2. 


Potato  inoculated  from  a  cultivation  in  nutrient  gelatine. 
In  three  days  a  peculiar  bluish-green  growth 
develops  on  the  surface  of  the  potato,  and  in 
nine  days  it  has  a  heaped-up  margin  of  a  bluish- 
green  colour,  while  the  central  portion  has  turned 
almost  black. 


PLATE  2L 


Fig  1.  After  three  days  drowtk. 


2.  After  nine  days  growth.. 


POTATO   CULTIVATION    OF  BACILLUS    CYANO GENUS. 


H.Cnxkthank  Pi«.r: 


^.L6wia,36.G(WW  Street. 


Vlnaonl  Bnolc.i.Day  itSon.Uth. 


PLATE  XXII. 

Bacillus  of  septicaemia  of  mice. 

FIG.  i. 

From  a  section  of  a  kidney  of  a  mouse  which  had  died 
after  inoculation  with  a  pure  cultivation  of  the 
bacillus.  With  moderate  amplification,  the  white 
blood-corpuscles  have  a  granular  appearance,  and 
irregular  granular  masses  scattered  between  the 
kidney  tubules  are  seen.  Stained  with  Gram's 
method  and  eosin.  x  200. 

FIG.  2. 

Part  of  the  same  preparation  with  high  amplification. 
The  granular  appearances  are  found  to  be  due 
to  the  presence  of  great  numbers  of  extremely 
minute  bacilli.  x  1,500. 


PLATE    22. 


.t.  Front  a  .section  of  Jtidney  of  &  mouse.. 
Gram's  method  a>nd£osin.  Zeiss '  DD.  Oc.  Z. 


\ 


BACILLUS     OF    SEPTIC.SMIA    OF   MICE. 

Fig.  Z.    The  sc^m-e  preparation/.  Xeiss'lk.  o.i.  Oc.  4. 


LdnaoivPublishedby  H.K.Lewi8.136.Gwwr Street. 


HnetntSrtttt^Day  i  Son..  lid.. 


PLATE  XXIII. 

Bacillus  leprse. 
FIG  i. 

From  a  section  of  the  skin  of  a  leper.  The  section 
is,  almost  in  its  entirety,  stained  purple,  and, 
with  moderate  amplification,  has  a  finely  granular 
appearance.  Stained  with  Ehrlich's  method 
(fuchsine  and  methylene  blue).  x  200. 

FIG.  2. 

Part  of  the  same  preparation  with  high  amplification, 
showing  that  the  appearances  described  above 
are  due  entirely  to  an  invasion  of  the  tissue  by 
the  bacilli  of  leprosy.  x  1,500. 


FUJ.  I.    From   a.  section  or  skin,  from  a.  case-  of  If.prosif. 
£hriich's  method.  (&ufasi/ne,  a-ncL  metfvyl&ie  Hue.)     ZCeiss  AA.  Oc. 


,-   •    r-.  '-fJ7w     rn     4 


BACILLUS        L 

Th,e  same-  pr&paraiiow.-steiss '  18.0.  i.   Oc. 4-. 


Edgt 


Street. 


PLATE  XXIV. 

FIG.  i. — Bacterium  lineola. 

Tube  inoculated  from  putrid  blood-serum  which 
swarmed  with  Bacterium  lineola.  The  same 
bacteria  were  found  in  the  cultivation. 

FIG.  2. — Micrococcus  rosaceus. 

Tube  inoculated  from  an  old  cultivation  on  nutrient 
agar-agar  which  had  become  contaminated.  The 
cultivation,  macroscopically  resembling  pink  yeast, 
consisted  of  a  pure  cultivation  of  micrococci. 

FIG.  3. — Streptococcus  pyogenes  citreus. 

Tube  inoculated  with  cocci  isolated  from  a  subcu- 
taneous abscess  in  a  mouse. 


CULTIVATIONS  ON  NUTRIENT  ACAR-AGAR 

Fitf  1.  Bacterium  lineola. 

Fig  2. Micro co ecus  rosaceus . 

Fitf  S.Staphjdococcus  pyo^enes  citreus. 


,  tf^ 


PLATE  XXV. 

Bacillus  figurans. 

Bacillus  mycoidcs. 
Wurzcl  Bacillus. 
FIG.  i. 

From  an  impression-preparation  of  a  growth  on  the 
surface  of  nutrient  gelatine.  x  50. 

FIG.  2. 

Part  of  the  same  preparation  with  high  amplification, 
showing  that  the  coils  and  filaments  of  the 
growth  are  due  to  a  peculiar  and  regular  arrange- 
ment of  the  individual  bacilli,  x  1,500. 


PLATE  25. 


Fig.  /.     Cover -glass  impre*swn -preparatums 
Zeiss'AA.Oc.  Z 


BACILLUS     FIGURANS. 

Fig.%.   The-  scutte  preparation.      Ze>i*ss' fa.   o..i.    Oc.   4-. 

Zdg or Cwkskank  tic.etpwt.  LondOIiPotiisKedby  H.K.Le"Ads,l3€,Gower  Street.  T>%nuntBa><*f,J)aytSon,  IiA. 


PLATE    XXVI. 

Bacillus  of  swine-erysipelas. 

Bacillus  oj   German  swine-fever. 
FIG.  i. 

Pure  cultivation  in  nutrient  gelatine.  The  growth  is 
stated  to  be  identical  with  that  of  the  bacillus  of 
septicaemia  in  mice. 

FIG.   2. 

Colonies  on  a  plate-cultivation. 

FIG.  3. 

Cover-glass  preparation  of  blood  from  an  inoculated 
pigeon. 

(After  Schiitz,  Arb.  a.  d.  Kais.  Gesuiidh.  A  nit,  Bd.  I.) 


PLATE   26. 


*-%.  \ 

v 

t^*— «=> 


XI 


BACILLUS    OF     SWINE-FEVER  (  S  CHUTZ  ). 

Fig.  I.     Pure  -  cufcwcdunv  in,  Ttu^rieiii  gelatine . 

Fig.  Z.     Colonies  on  a,  pla,te  -  cullvvcbtion, . 

fig.3 .    Cover -glass  f>  reparation  of*  blood,  from  an  inoculated  piqeon 

4 fur  SMitg  Arl.  a.  d,.Aa,istrl.  Geswid&tittsan&M  I.  LondoixRjbliihed  by  H .  K.  Lewis  ,136,  Cover  S  treet . 


PLATE  XXVII. 

Actinomyces. 
FIG.  i. 

From  a  section  of  a  maxillary  tumour  in  a  cow. 
Stained  by  Plaut's  method  (magenta  and  picric 
acid).  x  90. 

FIG.    2. 

Part  of  the  same  preparation,  with  higher  amplifica- 
tion. The  fungoid  masses  are  very  deeply  stained 
by  this  method.  The  component  club-shaped 
elements  and  their  radiate  arrangement  are  clearly 
shown.  x  500. 


PLATE   27. 


Fig.  I .   From  a.  faction/  of  a,  maxMa,ry  iM/nu>ur  in  a,  eow. 
fiaut's  -method  (Magenta,  ami  picric  a,ad).  7,  •/.»-.  i '  AA.  Vc.  4-. 


ACTINOMYCE  S. 

Fig.%.    The  same,  preparation.     Z&ijs'tk:  o.i.  Oc.  2. 


Londo.n.Rjblujhcdl     H .K.Le-A^G,  136, Gaver  Street . 


PLATE  XXVIII. 

Actinomyces. 
FIG.  i. 

From  a  section  of  a  maxillary  tumour  in  a  cow. 
Stained  by  Weigert's  method  (orseille  and  gen- 
tian violet),  x  900. 

FIG.  2. 

From  a  section  of  the  lung  of  a  cow.  The  rosettes  are 
much  smaller,  possibly  owing  to  their  being  more 
confined  by  their  surroundings  than  when  grow- 
ing in  the  soft  pulpy  tissue  of  the  maxillary 
tumour.  They  are  here  shown  with  high 
amplification,  but  under  a  power  of  about  50 
diam.  (Zeiss  A.A.  Oc.  2)  the  section  of  a  lung  re- 
sembles miliary  tuberculosis,  and  in  the  centre 
of  a  neoplasm  the  rosette  appears  about  the 
size  of  a  pin's  head.  Stained  with  Weigert's 
method  (orseille  and  gentian  violet).  x  500. 


PLATE     28 . 


tig.  J.   From,  a,  section-  of  a.  mcuc-iLLarii  tu^moiir   in,  a,  cotu .  - 
Wei<j&rts  method.  (OrsnilLe  and  gentian-violet).  Zeiys'ik.  o.  i.  Oc.  4-. 


ACTINOMYCE  S. 

Fig.  2.  From,  et  section,  of  the  lung  of  a,  cow. 
Weigert's  method,  (Orse,Me  and  gentian-violet.)  Zvifs'&.o.i.  Oc.Z. 


Edgar  CnoksKonk  hc.etpim 


London.rutit/Hedbv  H.K.LfrA-i^.i.-;0."-.  -er  Streel . 


PLATE  XXIX. 

FIGS,  i  to  ii. 

Yeast-fungi  or  saccharomycetes  and  mould 
fungi  or  hyphomycetes. 

(Facing  page   339.) 

For  description  see  p.  339. 


Plate    2'.}. 


YEAST-FUNGI  OR  SAGCHARDMXCETES  AND  MOULD-FUNGI  OR   HYPHOMTF.TKS. 


• 


BACTERIOLOGY. 

% 

CHAPTER    I. 

INI  ROD  UCTOR  Y. 

THE  researches  of  Pasteur  into  the  role  played  by 
micro-organisms  in  the  processes  of  fermentation 
and  putrefaction,  and  in  diseases  such  as  anthrax, 
the  silkworm  malady,  pyaemia,  septicaemia,  and 
chicken  cholera,  have  invested  the  science  of 
Bacteriology  with  universal  interest  and  vast 
importance.  The  further  researches  of  the  prac- 
tical mind  of  Lister,  with  the  resulting  evolution 
of  antiseptic  surgery,  have  demonstrated  the 
necessity  for  a  more  intimate  acquaintance  with 
the  life-history  of  these  micro-organisms;  while 
the  more  recent  investigations  which  have 
established  the  intimate  connection  between 
bacteria  and  infectious  diseases,  and  more  espe- 
cially the  discovery  by  Koch  of  the  tubercle  and 
cholera  bacilli,  have  claimed  the  attention  of 
the  whole  thinking  world. 

The   scientific    importance   of  these   latter   dis- 


2  BACTERIOLOGY. 

coveries  is  evidenced  by  the  fact  that  in  Germany 
medical  men  were  summoned  from  all  parts  of 
the  country  to  Berlin  to  attend  a  course  of 
instruction  in  the  latest  methods  employed  in  the 
investigation  of  bacteria.  In  this  way  the  methods 
of  pure  cultivation  devised  by  Koch,  and  the  means 
of  recognising  the  cholera  bacillus,  are  being  widely 
disseminated. 

To  a  medical  man,  those  bacteria  which  are  con- 
nected with  disease,  and  more  especially  those  which 
have  been  proved  to  be  the  causa,  if  not  the  actual 
materies  morbi,  are  of  predominant  interest  and  im- 
portance. It  is,  however,  impossible  by  localising 
one's  knowledge  to  pathogenic  species  to  thoroughly 
understand  the  life-history  of  these  particular 
forms,  or  to  be  able  to  grasp  and  appreciate 
the  various  arguments  and  questions  that  arise 
in  comparing  their  life-history  with  the  progress 
of  disease. 

It  is  not  sufficient  to  know  only  how  to 
recognise  and  artificially  cultivate  a  bacterium 
associated  with  disease  ;  we  must  endeavour  to 
establish  the  exact  relationship  of  the  bacterium 
to  the  disease  in  question.  To  ascertain  beyond 
all  doubt  whether  a  micro-organism  is  actually 
the  causa  causans  of  a  disease,  Koch  has  laid 
down  the  following  postulates  : — 

a.  The  micro-organism  must  be  found  in  the 
blood,  lymph,  or  diseased  tissues  of  man,  or  animal, 
suffering  from,  or  dead  of,  the  disease. 


INTRODUCTORY.  3 

b.  The  micro-organisms  must   be  isolated   from 
the  blood,  lymph,  or  tissues,  and  cultivated  in  suit- 
able media, — i.e.,  outside  the  animal  body.     These 
pure  cultivations  must  be  carried  on  through  succes- 
sive generations  of  the  organism. 

c.  A  pure-cultivation  thus  obtained  must,  when 
introduced  into  the  body  of  a  healthy  animal,  pro- 
duce the  disease  in  question. 

d.  Lastly,   in    the  inoculated   animal   the    same 
micro-organism  must  again  be  found. 

These  points  would  naturally  suggest  a  sequence 
in  the  various  processes  which  must  be  adopted  in 
a  practical  study  of  micro-organisms  associated  with 
disease.  Inasmuch,  however,  as  these  processes 
embrace  those  which  are  employed  in  the  isolation, 
cultivation,  etc.,  of  non-pathogenic  species,  we  shall, 
in  studying  the  bacteria  as  a  whole,  adopt  the  order 
suggested.  After  an  introduction  to  the  apparatus 
commonly  employed  in  a  bacteriological  laboratory, 
we  shall  describe  the  methods  of  examining  liquids, 
tissues,  etc.,  and  the  means  of  recognising  micro- 
organisms. Then  will  follow  the  methods  of 
isolating  these  micro-organisms  from  such  liquids, 
tissues,  etc.,  and  of  carrying  on  pure  cultivations 
in  nutrient  media.  Lastly,  we  shall  refer  briefly 
to  experimental  researches  on  the  living  animal, 
and  the  means  of  isolating  micro-organisms  from  the 
liquids  and  tissues  of  the  body  after  death. 

In  Part  II.  will  be  found  chapters  upon  the 
General  Biology  of  bacteria,  and  in  Part  III.  a 


4  BACTERIOLOGY. 

chapter  upon  their  classification,  followed  by  a  de- 
scription of  each  species,  more  particularly  of  those 
of  pathological  interest,  with  a  detailed  account  of 
the  special  methods  of  examination  and  of  staining 
employed  for  demonstrating  the  different  species. 

In  the  Appendix  a  descriptive  list  of  important 
yeasts  and  moulds  will  be  given,  with  any  special 
technique  required  in  their  case.  Yeasts  and 
moulds  are  constantly  encountered  in  the  special 
methods  for  examining  bacteria  in  air,  soil,  and 
water,  and  several  are  of  interest  in  being,  like 
many  bacteria,  micro-organisms  associated  with 
disease.  A  short  account  is  also  given  of  the 
Flagellated  Protozoa,  which  have  been  found  to 
occur  in  the  blood  of  animals.  To  the  pathologist 
these  are  of  interest  owing,  more  especially,  to  the 
discovery  of  closely  allied  micro-parasites  in  the 
blood  in  cases  of  malaria. 

The  special  methods  just  referred  to,  with 
description  of  the  apparatus  employed,  and  a 
Chronological  Bibliography,  are  also  comprised  in 
the  Appendix. 


CHAPTER  II. 

APPARATUS,  MATERIAL,  AND  REAGENTS 
EMPLOYED  IN  A  BACTERIOLOGICAL  LABORATORY 

(A)     HISTOLOGICAL    APPARATUS. 

Microscope. — For  the  investigation  of  micro- 
organisms a  good  microscope  with  oil-immersion 
system  and  a  condenser,  such  as  Abbe's,  is 
essential.  Such  instruments  are  supplied  by 
Leitz,  Zeiss,  and  Hartnack  in  Germany,  and 
Powell  and  Lealand  in  England.  Zeiss'  micro- 
scope, with  -fj  and  T*g-  oil-immersion  lenses,  or 
Powell  and  Lealand's  with  •£%  and  •£$,  is  recom- 
mended for  investigators;  while  Leitz',  with  r^-,  is 
a  serviceable  and  economical  one  for  students.*  In 
addition  to  the  usual  microscopic  fittings,  Zeiss 
supplies  a  micrometer  eyepiece,  with  directions 
for  use.  Some  such  arrangement  is  essential  for 
the  measurement  of  bacteria.  Other  accessories  to 
the  microscope  are — 

A  large  bell-glass  for  covering  the  microscope  when  not 
in  use. 

About  a  foot  square  of  blackened  plate-glass. 

*  Leitz'    with  ^  costs  about  ^15  ;    Zeiss',  with  the  same, 
and  with  -fa,  ;£2°  more.     Refer  to  foot-note  on  p.  44. 


0  BACTERIOLOGY. 

A  white  porcelain  slab  of  the  same  size. 

Glass  bottles  with  ground  glass  stoppers,  for  alcoholic 
solutions  of  aniline  dyes,  etc. 

Glass  bottles  with  funnels,  for  aqueous  solutions  of  the 
dyes,  and  others  provided  with  pipettes. 

A  small  rod-stoppered  bottle  of  cedar  oil.  This  is 
recommended  by  Zeiss  in  preference  to  other  oils  for  his 
immersion  lenses. 

Set  of  small  glass  dishes  or  capsules,  and  watch  glasses, 
for  section  staining,  etc. 

Stock  of  best  glass  slides,  in  packets  of  fifty. 

Several  boxes  of  round  and  square  thin  cover-glasses,  in 
various  sizes,  of  the  best  quality. 

Needle-holders,  with  a  couple  of  platinum  needles,  and  a 
packet  of  ordinary  sewing  needles. 

Glass  rods  drawn  out  to  a  fine  point ;  useful  for  manipu- 
lating sections  when  acids  are  employed. 

Copper  lifters,  preferably  plated. 

One  pair  of  small  brass  or  spring-steel  platinum-pointed 
forceps,  for  holding  cover-glasses. 

One  pair  of  brass  tongs. 

Collapsible  tubes  for  containing  Canada-balsam  ;  very 
serviceable  for  transport  and  general  use. 

Turn-table,  used  in  preparing  slides  with  rings,  for 
mounting  preparations  of  Aspergillus,  etc. 

Boxes  for  preparations,  book-form. 

Tickets  and  labels,  various  sizes, 

Soft  rags  or  old  pocket  handkerchiefs,  for  removing  cedar 
oil  after  use  of  immersion  lens,  cleaning  cover-glasses,  etc. 

Chamois  leather  for  wiping  lenses. 

Microtome. — Schanze's  is  much  in  favour  in 
Germany,  but  Jung's,  of  Heidelberg,*  though  a 

*  Price  lists  may  be  obtained  from  any  of  the  above-mentioned 
firms,  from  which  an  idea  of  the  instruments  can  be  formed,  and  a 
comparison  of  the  prices  made. 


APPARATUS,    MATERIAL,    AND    REAGENTS.  7 

somewhat  cumbrous  instrument,  is  much  to  be 
preferred.  Smaller  accessories,  which  should  be 
within  reach,  are — 

A  small  can  of  sewing  machine  oil. 

A  soft  rag  and  chamois  leather,  for  wiping  the  knives 
immediately  after  use. 

Stone  and  leather  for  setting  and  sharpening  the  same. 
Two  or  three  camel's  hair  brushes. 

A  Freezing  Microtome,  such  as  Williams'  or 
Roy's,  and  a  Valentin's  Knife,  are  useful  for  the 
examination  of  tissues  in  the  fresh  state,  but  other- 
wise are  supplanted  by  the  above. 

(B)  REAGENTS  AND  MATERIAL  EMPLOYED  IN  THE 
PROCESSES  OF  HARDENING,  DECALCIFYING,  EM- 
BEDDING, FIXING,  AND  CUTTING  OF  TISSUES. 

Alcohol,  absolute. 
Bergamot  oil. 
Celloidin. 

Dissolved  in  equal  parts  of  ether  and  alcohol. 

Cork,  or  stock  of  ready-cut  corks. 

Kbner's  solution.  A  mixture  in  the  following 
proportions : — 

Hydrochloric  acid         .          .          .  5- 

Alcohol        ...  .          100 

Distilled  water     .  .20 

Chloride  of  sodium        ...  5' 


8  BACTERIOLOGY. 

Gelatine. 

Melted  in  a  small  porcelain   capsule  and  set 
aside  ready  to  be  re-melted  when  required  for  use. 

Glycerine-gelatine  (Klebs). 

Best  well  washed  gelatine         .         .        10 
Add  distilled  water,  allow  gelatine 
to   swell   up,    pour   off  excess  of 
water,   melt  gelatine  with  gentle 
heat,  add 

Glycerine         .         .         .         .         .10 
Lastly  a  few  drops  of  phenol  for  preservation 

Gum. 

Kleinenberg's  solution. 

Saturated  watery  solution  of  picric 

acid     ......     100 

Strong  sulphuric  acid       ...         2 
Filter  and  add 

Distilled  water         ....     300 

Muller's  fluid. 

Bichromate  of  potash  .         .         2 

Sulphate  of  sodium  i 

Distilled  water         .         .         .         .100 

Osmic  acid. 

Distilled  water         .         ,         .         .100 
Osmic  acid       .         .         .         .         .  '5 


APPARATUS,  MATERIAL,  AND  REAGENTS.     9 

Paper  trays. 
Paraffin. 
Spermaceti. 
Xylol. 

Hardening  and  decalcifying  solutions  should  be 
kept  in  stock  in  quantities  according  to  require- 
ment. A  jacket  of  brown  paper  should  be  pasted 
round  a  well  stoppered  bottle  to  contain  osmic  acid 
to  efficiently  protect  it  from  light,  and  it  should  be 
kept  in  a  cool  place.  The  celloidin  solution  may  be 
kept  in  stock  in  a  wide-mouthed  glass  bottle,  from 
which  small  wide-mouthed  bottles  may  be  filled 
according  to  the  number  required.  To  put  several 
pieces  of  different  tissues  in  the  same  bottle  leads 
to  confusion. 

(C)      REAGENTS      FOR     EXAMINING     AND      STAINING 
MICROSCOPICAL    PREPARATIONS. 

1.  Acetic  acid,  strong. 

2.  Alcohol — absolute. 

3.  Alcohol — 60  per  cent. 

4.  Alcohol — acidulated. 

Alcohol  .         .         .         .  .100 

Hydrochloric  acid    .         .         .         .          i 


IO  BACTERIOLOGY. 

5.  Alum  carmine  (Grenadier). 

Carmine .          .  •         •         •         i 

Five  per  cent,  solution  of  alum         .     100 

Boil  twenty  minutes,  filter  when  cold. 

6.  Ammonia,  strong. 

7.  Aniline. 

8.  Aniline  water. 

Distilled  water          .         ,         .         .100 

Aniline    .         .         .  •         5 

Shake  well  and  filter  emulsion. 

9.  Bismarck  brown. 

(a)  Concentrated  solution  in  equal  parts  of  gly- 
cerine and  water. 

(,')  Aqueous  solution. 

Bismarck  brown       .         .         .         •         2 
Alcohol  ...         .  15 

Distilled  water  •       85 

10.  Borax  carmine  (Grenacher). 

Borax      .         .         .         .  .          2 

Carmine          ...  .        '5 

Distilled  water          .          .          .          .100 

To  the  dark  purple  solution  add  a  5  p.  c.  sol.  of 
acetic  acid  until  a  red  colour  is  produced ;  set  aside 
24  hours,  filter,  and  add  a  drop  of  carbolic  acid. 


APPARATUS,    MATERIAL,    AND    REAGENTS. 

11.  Cedar  oil. 

12.  Eosin. 

(a)  Saturated  alcoholic  solution. 

(b)  Aqueous  solution. 

Distilled  water         .         .       ,  .         .     TOO 
Eosin       .  ...         5 

13.  Ether. 

14.  Fuchsine. 

(a)  Saturated  alcoholic  solution. 

(b)  Aqueous  solution. 

Fuchsine           .....         2 
Alcohol  .         .         .         .         .  15 

Water 85 

15.  Gentian  violet. 

(a)  Saturated  alcoholic  solution. 

(b)  Aqueous  solution. 

Gentian  violet          ,  .         .         2^25 

Distilled  water         .         .         .         .100 

1 6.  Gibbes'  solution,  for  double  staining. 

Take  of 

Rosaniline  hydrochlorate .       .  .         .         2 
Methylene  blue  i 

Triturate  in  a  glass  mortar, 


T  2  BACTERIOLOGY. 

Dissolve  aniline  oil .  ,  .  ,  3 
In  rectified  spirit  .  .  .  1 5 

and  add  slowly  to  the  above. 

Lastly,  slowly  add  distilled  water      .       15 
Keep  in  stoppered  bottle. 

17.  Glycerine,  pure. 

1 8.  Haematoxylin  solution. 

Hsematoxylin  .....         2 

Alcohol  .         .         .         .         .         .     100 

Distilled  water        '.       ".  "      .         .      100 
Glycerine         .         .         .         .         .100 

Alum       .         .        .         .         .         .2 

19.  Iodine  solution. 

Iodine,  pure  .....  i 
Iodide  of  potassium  .,  .  • .  "  2 
Distilled  water  ....  50 

20.  Iodine  solution  (Gram). 

Iodine     ......          i 

Iodide  of  potassium  .  .  .  2 
Distilled  water  ....  300 

21.  Lithium-carmine  solution  (Orth). 

Saturated  solution  of  carbonate  of 

lithium      .         .         .         .         .100 
Carmine         ,  , ,  .         .         .         2*5 


APPARATUS,    MATERIAL,    AND    REAGENTS.          13 

22.  Magenta  solution  (Gibbes), 

Magenta          .         .  .                  .  2 

Aniline  oil                .  .         .         .  3 

Alcohol  (Sp.  Gr.  '830)  ...  20 

Distilled  water.         .  .         .         .  20 

23.  Methylene  blue. 

(a)  Concentrated  alcoholic  solution. 

(ft)  Aqueous  solution. 

Methylene  blue  ....  2 
Alcohol  .  .  .  .  .  -15 
Water 85 

(c)  Koch's  solution. 

Concentrated  alcoholic  solution  of 

methylene  blue  i 

Ten  per  cent,  potash  solution  .         .  2 

Distilled  water         ....  200 

(a}  Loffler's  solution. 

Concentrated  alcoholic  solution  of 

methylene  blue     ....       30 
Solution  of  potash  i — 10,000  .         .100 

24.  Methyl  violet. 

(a)  Concentrated  alcoholic  solution. 


14  BACTERIOLOGY. 

(b)  Aqueous  solution. 

Methyl  violet  .         .         .         .         2.25 

Distilled  water         .         •         •         «     100 

(c)  Koch's  solution. 

Aniline  water  .         .         .         .         .100 
Alcoholic  solution  of  methyl  violet  1 1 

Absolute  alcohol      .         .         .         .10 

25.  Neelsen's  solution. 

Dissolve  fuchsine  i 

In  a  5  per  cent,  watery  solution  of 

carbolic  acid         ...         .  .100 

Add  alcohol    .         .         .         .  .10 

26.  Nitric  acid,  pure. 

27.  Orseille  (Wedl). 

Dissolve  pure  ammonia-free  orseille  in 
Absolute  alcohol       ...       20 
Acetic  acid       .         .         .         .         5 
Distilled  water ....       40 
until  a  dark  red  liquid  results  :  filter. 

28.  Picric  acid. 

(a)  Concentrated  alcoholic  solution. 

(b)  Saturated  aqueous  solution. 


APPARATUS,    MATERIAL,    AND    REAGENTS.  15 

29.  Picro-carmine  (Ranvier). 

Carmine            .....  i 

Distilled  water         .         .         .         .  10 

Solution  of  ammonia        .         .         ,  3 
Triturate,  add  cold  saturated  solution 

of  picric  acid        ....  200 

30.  Picro-lithium-carmine  (Orth). 

To  above  mentioned  Lithium-carmine 

solution  add 
Saturated  solution  of  picric  acid       .       2*3 

31.  Potash  solution. 
(a)  I   to  3  per  cent. 
(£)  10     „     „ 

W     -     33     i,     „ 

32.  Safranine. 

(a)  Concentrated   alcoholic  solution. 
(6)  Watery  solution      .      i  per  cent. 

33.  Sulphuric  acid,  pure. 

34.  Salt  solution        .        0*8  per  cent. 

35.  Turpentine. 

36.  Vesuvin. 

(a)  Concentrated  alcoholic  solution. 

(b)  Watery  solution. 


1 6  BACTERIOLOGY. 

Water,  distilled. 

Water,  sterilised. 

Distilled  water  can  be  kept  for  use  in  a  wash 
bottle,  or  far  better  in  a  siphon  apparatus.  Steri- 
lised water  is  convenient  in  plugged  sterile  test-tubes 
which  may  be  kept  close  at  hand  in  a  beaker, 
or  tumbler,  with  a  pad  of  cotton  wool  at  the 
bottom.  The  numbered  reagents  can  be  conveni- 
ently arranged  on  shelves  within  easy  reach. 
Alcoholic  solutions  of  the  aniline  dyes  and  other 
special  preparations  should  be  kept  in  bottles 
with  ground  glass  stoppers.  Aqueous  solutions  of 
the  dyes  may  be  kept  in  bottles  with  funnel  filters, 
and  the  solution  filtered  before  use.  To  both 
aqueous  and  alcoholic  solutions  a  few  drops  of 
phenol,  or  a  crystal  of  thymol,  should  be  added  as 
a  preservative.  For  the  rapid  staining  of  cover- 
glass  preparations,  it  is  convenient  also  to  have  the 
most  frequently  used  stains  (fuchsine,  methyl-violet) 
in  bottles  provided  with  pipette-stoppers. 

(D)    REAGENTS     FOR    MOUNTING    AND     PRESERVING 
PREPARATIONS. 

Acetate  of  potash. 

Concentrated  solution. 

Asphalte  lac. 


APPARATUS,  MATERIAL,  AND  REAGENTS.    I? 

Canada  balsam. 
Dissolved  in  xylol. 

Glycerine  gum  (Farrant's  solution). 
Glycerine. 
Water. 

Saturated  solution  of  arsenious  acid. 
Equal  parts,  mix  and  add  of  picked  gum  arable 
half  a  part. 

Hollis'  glue. 
Zinc-white. 

(E)    DRAWING    AND    PHOTOGRAPHIC   APPARATUS. 

Camera  Lucida. — The  camera  lucida  of  Zeiss 
is  an  excellent  instrument,  though  many  prefer  the 
pattern  made  by  Nachet  of  Paris.  Combined  with 
the  use  of  a  micro- millimeter  objective  it  affords  also 
a  simple  method  for  the  measurement  of  bacteria. 

For  drawing  macroscopical  appearances,  and  for 
illustrating  microscopical  specimens  with  or  without 
the  use  of  a  camera  lucida,  the  following  materials 
should  be  within  reach  : — 

Pencils. 
Etching  Pens. 
Prepared  Indian  Ink. 
Water-colour  Paints  and  Brushes. 
Ordinary  and  tinted  drawing  paper  and  other  usual 
accessories. 

Photo-micrographic  Apparatus.  —  Zeiss  of 
Jena,  Seibert  &  Kraft  of  Wetzlar,  Nachet  of  Paris, 

2 


1 8  BACTERIOLOGY. 

and  Swift  &  Son  of  London,  may  all  be  recom- 
mended for  constructing  an  arrangement  in  which 
the  photographic  camera  is  combined  with  the 
microscope. 

For  illumination  either  sunlight  or  artificial  light 
may  be  employed.  In  the  case  of  sunlight  a  helio- 
stat  is  necessary  to  procure  the  best  results,  but  as 
sunlight  is  not  always  available  by  day,  and  it  is 
also  more  convenient  for  many  to  work  at  night,  it 
is  better  to  have  recourse  altogether  to  artificial 
light.  Excellent  results  may  be  obtained  with  an 
ordinary  paraffine  lamp,  or  with  magnesium,  oxycal- 
cium,  or  electric  light.  Specimens  are  preferably 
stained  yellow,  brown,  or  black,  and  for  mounting 
the  preparations  Koch*  recommends  a  saturated 
solution  of  acetate  of  potash  ;  but  there  is  little  or 
no  objection  to  the  use  of  Canada  balsam  dissolved 
in  xylol.  Hauser,f  who  employed  the  electric  light, 
obtained  some  excellent  pictures  of  preparations 
mounted  in  balsam.  Van  Ermengem  J  first  recom- 
mended the  isochromatic  dry  plates,  and  produced 
most  successful  results  with  the  lime-light  from 
objects  stained  with  fuchsine  and  methyl-violet 
The  author  also  has  investigated  the  applicability 
of  photographic  processes  for  illustrating  micro- 

*  Koch,  Verfahren  zur  Untersuchung  zum  Conserviren  und 
Photograph  iren  der  Bacterien .  1877. 

t  Hauser,  Uber  Faulniss  Bacterien  und  deren  Beziehungen  zur 
Septicamie.  1885. 

\  Van  Ermengem,  Bull,  de  la  Soc.  Beige  de  Microscope,  No.  X., 
pp.  170-2.  1884. 


APPARATUS,    MATERIAL,    AND    REAGENTS.          1 9 

organisms.  Numerous  preparations  have  been 
satisfactorily  depicted  by  means  of  the  isochromatic 
plates  without  any  reference  to  the  staining  reagents 
employed.  For  a  full  description  of  the  apparatus 
and  methods  employed  the  reader  is  referred  to 
the  author's  publication.* 


(F)    STERILISATION    APPARATUS. 

Steam-Steriliser. — A  cylindrical  vessel  of  tin 
about   half  a   metre    or    more   in 
height,  jacketed    with   thick   felt, 
and  provided  with  a   conical   cap 
or  lid  (Fig.   i).      The   lid  is   also 
covered  with  felt,  has  handles  on 
either   side,  and   is   perforated   at 
the  apex  to  receive  a  thermometer. 
Inside  the  vessel  is  an  iron  grating 
or  diaphragm  about  two-thirds  the 
way  down,  which  divides  the  interior 
into  two  chambers — the  upper  or 
"steam  chamber,"  and  the  lower 
or    "  water-chamber."      A  gauge 
outside  marks  the  level  of  the  water      FIG.  i.— KOCH'S 
in  the  lower  chamber;  this  should 
be    kept    about    two-thirds    full.     The    apparatus 
stands  upon  three  legs,  and  is  heated  from  below 
with  two  or  three  Bunsen,  or  better,  a  Fletcher's 

*  Photography  of  Bacteria.     1887. 


BACTERIOLOGY. 


burner.     It  is   employed    for    sterilising    nutrient 

media  in  tubes  or  flasks, 
for  cooking  potatoes,  or 
hastening  the  filtration 
of  agar  -  agar.  When 
the  thermometer  indi- 
cates 1 00°  C.  the  lid  is 
removed,  and  test-tubes 
are  lowered  in  a  wire 
basket  by  means  of  a 
hook  and  string,  and 
the  lid  quickly  replaced. 

FIG.  2. -HOT-AIR  STERILISER.       potatoes    Or  small   flasks 

are  lowered  into  the  cylinder  in  a  tin  receiver  with 
a  perforated  bottom,  which  rests 
upon  the  grating  and  admits  of 
its    contents   being  -exposed   to 
the  steam. 

Hot-air  Steriliser. — A  cubi- 
cal chest  of  sheet  iron  with 
double  walls,  supported  on  four 
le^js ;  it  may  also  be  suspended 
on  the  wall  of  the  laboratory, 
with  a  sheet  of  asbestos  inter- 
vening (Figs.  2  and  3).  It  is 
heated  with  a  rose  gas-burner 
from  below,  and  the  temperature 
of  the  interior  indicated  by  a 
thermometer  inserted  through 

i     i      .      .,  e  ,          FIG.  3. — SECTION 

a  hole  m  the  roof;  in  a  second  OF  HOT-AIR  STERILISER. 


APPARATUS,    MATERIAL,    AND    REAGENTS.          21 

opening  a  gas  regulator  can  be  fixed.  Test-tubes, 
flasks,  funnels,  cotton  wool,  etc.,  may  be  sterilised 
by  exposure  to  a  temperature  of  150°  C.  for  an 
hour  or  more. 


(G)  APPARATUS  AND  MATERIAL  FOR  PREPARING 
AND  STORING  GELATINE-,  AND  AGAR-AGAR- 
PEPTONE-BROTH. 

Water-bath. — A  water-bath  on  tripod  stand 
is  required  for  boiling  the  ingredients  of  nutrient 
jellies  and  for  general  purposes.  The  lid  may 
be  conveniently  composed  of  a  series  of  con- 
centric rings,  so  that  the  mouth  of  the  vessel 
may  be  graduated  to  any  size  required. 

Test-tube  Water-bath.-  —  This  consists  ot 
a  circular  rack  for  test-tubes  within  a  water- 
bath.  It  is  sometimes  employed  instead  of  the 
steam  cylinder  for  sterilising  nutrient  jelly  in 
tubes  by  boiling  for  an  hour,  for  three  successive 
days. 

Hot-water  Filter. — A  copper  funnel  with 
double  walls,  the  interspace  between  which  is 
filled  with  hot  water.  A  glass  funnel  fits  inside 
the  copper  cone,  the  stem  of  the  glass  funnel 
passing  through  and  being  tightly  gripped  by  a 
perforated  caoutchouc  plug,  which  fits  in  the 
opening  at  the  apex  of  the  cone.  The  water  in 
the  cone  is  heated  by  applying  the  flame  of  a 


22  BACTERIOLOGY. 

burner  to  a  tubular  prolongation  of  the  water 
chamber.  In  a  more  recent  model,  as  repre- 
sented in  Fig.  4,  this 
prolongation  is  dispensed 
with,  and  the  tempera- 
ture is  maintained  by 
means  of  a  circular 
burner  which  acts  at  the 
same  time  as  a  funnel 
ring. 

Glass  Vessels.  —  A 
number  of  glass  vessels 
should  be  kept  in  stock 
according  to  require- 
ments. 

Bohemian  hard  glass 
flasks  are  employed  in 
several  sizes,  for  boil- 
ing- nutrient  media.  The 

FIG.  4.  a  t 

HOT- WATER  FILTERING  APPARATUS   Conical      forms       are       CS- 
WITH  RING  BURNER.  .   1f  ,  ., 

pecially  used  in  the 
larger  sizes  for  storing  nutrient  jelly. 

Glass  funnels  large  and  small  are  necessary, 
not  only  in  the  processes  of  preparing  nutrient 
jelly,  but  for  filtering  solutions  of  aniline  dyes 
and  for  general  purposes. 

A  liberal  supply  of  test-tubes  should  always  be 
kept  in  stock,  as  they  are  not  only  employed  for 
the  tube-cultivations,  but  can  be  conveniently 
used  for  storing  bouillon,  sterilised  water,  etc. 


APPARATUS,    MATERIAL,    AND    REAGENTS.          23 

Cylindrical  glasses  graduated  in  cubic  cen- 
timetres, 10  ccm.,  100  ccm.,  500  ccm.,  are 
required  for  measuring  the  liquid  ingredients  of 
nutrient  jelly,  and  also  in  preparing  the  various 
staining  solutions. 

A  large  wide- mouthed  glass  jar,  with  a  glass 
cover,  is  extremely  useful.  It  must  be  padded 
at  the  bottom  with  cotton  wool  for  containing  a 
stock  of  tubes  of  sterilised  nutrient  jelly,  and 
should  be  placed  within  reach  on  the  working 
table. 

Balance  and  Weights.  —  A  balance,  with 
large  pans  and  set  of  gramme  weights,  is  con- 
stantly required. 

Cotton  Wool.— The  best  or  "  medicated " 
cotton  wool  should  be  procured. 

Gelatine.  —  The  gelatine  for  bacteriological 
purposes  must  be  of  the  very  best  quality  (gold 
label). 

Agar-Agar. — This  is  also  called  Japanese  Isin- 
glass ;  it  consists  of  the  shrivelled  filaments  of 
certain  Algae  (Gracilaria  lichenoides  and  Gigartina 
speciosa).* 

Peptonum  Siccum  (Savory  and  Moore). 

Table  Salt. — Prepared  table  salt  can  be  ob- 
tained in  tins  or  packets. 

Litmus  Papers. — Blue  or  red  litmus  paper  in 
cheque  books,  for  testing  the  gelatine  mixture, 
etc. 

*  Ilueppe,  Die  Methoden  der  Bakterien  Forschung.  1885. 


24  BACTERIOLOGY. 

Carbonate  of  Soda. — A  bottle,  containing  a 
saturated  solution  of  carbonate  of  soda,  and 
provided  with  a  pipette-stopper,  may  be  kept, 
especially  for  use  in  the  preparation  of  nutrient 
jelly. 

Lactic  Acid. 

Filter  Paper. —  For  filtering  gelatine  stout 
Swedish  filter  paper  of  the  best  quality  is  re- 
commended. 

Flannel  or  Frieze. —  This  is  employed  as  a 
substitute  for,  or  combined  with,  filter  paper  in  the 
preparation  of  nutrient  agar-agar. 


(H)     APPARATUS     FOR     EMPLOYMENT     OF     NUTRIENT 
JELLY    IN     TEST-TUBE    AND    PLATE     CULTIVATIONS. 

Wire  Cages.  —  These  cages  or  crates  are 
used  for  containing  test-tubes,  especially  when 
they  are  to  be  sterilised  in  the 
hot-air  steriliser  ;  or  for  lowering 
tubes  of  nutrient  jelly  into  the 
steam  steriliser,  etc.  (Fig.  5). 

Test-tube   Stands.— The  or- 
dinary   wooden    pattern,    or    the 


metallic      folding      stands,      are 
FIG.  5.— WIRE  CAGE    called   into   use   for   holding  cul- 

FOR  TEST-TUBES.  .  _  ,  ' 

tivations.  Pegged  racks  are 
also  recommended  for  draining  test-tubes  after 
washing. 


APPARATUS,    MATERIAL,    AND    REAGENTS.          25 

Caoutchouc  Caps. — These  are  caps  for  fitting 
over  the  cotton  wool  plugs,  and  may  be  used  in 
different  sizes  for  test-tubes  and  stock-flasks. 

Platinum  Needles. — A  platinum  needle  for 
inoculating  nutrient  media,  examining  cultivations, 
etc.,  consists  of  two  or  three  inches  of  platinum 
wire,  fixed  to  the  end  of  a  glass  rod.  Several  of 
these  needles  should  be  made,  with  platinum  wire 
of  various  thicknesses.  A  piece  of  glass  rod, 
about  seven  inches  long,  is  heated  at  the  extreme 
point  in  the  flame  of  a  Bunsen  burner,  and  a  piece 


FIG.  6.— PLATINUM  NEEDLES. 

of  platinum  wire,  held  near  one  extremity  with 
forceps,  is  then  fused  into  the  end  of  the  rod. 
Some  needles  should  be  perfectly  straight,  and 
kept  especially  for  inoculating  test  -  tubes  of 
nutrient  jelly.  For  other  purposes  the  needles 
may  also  be  bent  at  the  extremity  into  a  small 
hook  or  a  loop*  (Fig.  6) 

Tripod  Levelling-stand  — A  triangular  wooden 
frame    supported     upon    three    screw-feet,    which 

*  A  looped  platinum  needle  is  called  in  Germany  an  "  ose,"  a  term 
which,  on  account  of  its  brevity,  may  be  conveniently  adopted 


26  BACTERIOLOGY. 

enable  it  to  be  raised  or  lowered  to  adjust  the 
level. 

Large  Glass  Plate. — A  piece  of  plate-glass, 
or  a  pane  of  ordinary  window  glass,  about  a  foot 
square. 

Spirit  Level. 

Glass  Bells  and  Dishes* — Shallow  glass  bells 


FIG.  7.— DAMP-CHAMBER  FOR  PLATE  CULTIVATIONS. 

and   dishes,    for   making   a   dozen  or   more  damp 
chambers   (Fig.    7),   and   for   completing   the  ap- 


FIG.  8.— APPARATUS  EMPLOYED  FOR  PLATE-CULTIVATIONS. 

Tripod  stand  ;  Glass  dish,  filled  with  cold  or  iced  water  ;  Sheet  of  Plate-glass  ; 
Spirit  Level,  and  Glass  Bell. 

paratus  for  pouring  out  liquefied  nutrient  jelly  on 
glass  plates  or  slides  (Fig.  8). 

Iron   Box. — A  box  of  sheet-iron    (Fig.   9)    for 


APPARATUS,    MATERIAL,    AND    REAGENTS.          2J 

containing  glass   plates   during  their   sterilisation 
in   the   hot-air    steriliser,    and    for 
storing    them    until    required    for  /I 
use. 

Glass     Plates. — Small     panes  '" 
of  glass,  about  six  inches  by  four. 
Not  less  than  three  dozen  are  re-  | 
quired  for  a  dozen  damp  chambers.  "• 

Glass    Benches. — These     are      BOX  FOR  GLASS 

f  -.11  PLATES. 

necessary  for  arranging  the  glass 
plates   or   slides  in    tiers    in    the   damp   chambers 
(Fig.  10).     Metal  shelves  may  be   substituted   for 
them,  but  the  former  are  to  be  preferred.     They 
can  be   easily  made,  in  any  number  required,  by 


FIGS.  10,  n.— GLASS  BENCHES  FOR  GLASS  PLATES  OR  SLIDES. 

cementing  a  little  piece  ot  plate  glass  at  either 
end  of  a  glass  slip  (Fig.  1 1). 

Glass  Rods. — One  dozen  or  more  glass  rods, 
twelve  to  eighteen  inches  in  length.  They  are 
employed  for  smoothly  spreading  out  the  liquefied 
nutrient  gelatine  or  agar-agar  on  the  glass  plates, 
etc. 

Thermometers.  —  Two  or  .three  centigrade 
thermometers. 


28 


BACTERIOLOGY. 


FIG.  12. — ISRAEL'S  CASE. 


(l)      APPARATUS     FOR     PREPARATION     OF     POTATO - 
CULTIVATIONS. 

Israel's  Case. — Sterilising   instruments   in   the 

flame  of  a  Bunsen  burner  is  most  destructive.     It 

is  better,  there- 
fore, to  have  a 
sheet -iron  case 
(Fig.  12)  to 
contain  potato- 
knives,  scalpels, 
and  other  instru- 
ments, and  to 

sterilise  them  by  placing  the  case  in   the  hot-air 

steriliser  for  an  hour  at  150°  C.     The  box  can  be 

opened  at  the  side,  and  each  instrument  withdrawn 

with  a  pair  of  sterilised  forceps  when  required  for  use. 
Glass    Dishes. — Several    shallow   glass   dishes 

are   required    for    preparing    damp    chambers    for 

potato       cultivations 

(Fig.  13).   The  upper, 

being  the  larger,  fits 

over   the  lower,  and 

having     no     handle, 

admits  of  these  damp 

chambers       being 

placed,  if  necessary, 

in  the  incubator  in  tiers.     The  large  size  may  also 

be  used  in  the  same  way  for  plate  cultivations. 
Potato    Knives. — A   common    broad   smooth  - 


FIG.  13. 

DAMP-CHAMBER  FOR  POTATO- 
CULTIVATIONS. 


APPARATUS,    MATERIAL,    AND    REAGENTS, 


29 


bladed  knife  set  in  a  wooden  handle  is  sold  for 
this  purpose. 

Scalpels.  —  Half  a  dozen  scalpels,  preferably 
with  metal  handles,  may  be  kept  especially  for 
inoculating  sterilised  potatoes. 

Brush. — A  common  stout  nail-brush,  or  small 
scrubbing  brush,  is  essential  for  cleansing  potatoes. 

(j)    APPARATUS    FOR    PREPARATION    OF    SOLIDIFIED 
STERILE    BLOOD    SERUM. 

Glass  Jar. — A  tall  cylindrical  glass  jar,  on  foot, 
with  a  broad  ground  stopper,  for  receiving  blood. 

Pipette. — An  ordinary  or  graduated  pipette  for 
transferring  the   serum 
from  the  jars  to  sterile 
test-tubes  or  glass  cap- 
sules. 

Serum-Steriliser. — 
A  cylindrical  case,  with 
double  walls  forming  an 
interspace  to  contain 
water,  closed  with  a  lid, 
also  double  walled  and 
provided  with  a  tubular 
prolongation  of  the  en- 
closed water  chamber 
(Fig.  14).  The  water 
in  the  cylinder  is  heated 
from  below,  and  that  in  the  lid  by  means  of  the 
prolongation. 


FIG.  14.— SERUM  STERILISER 


30  BACTERIOLOGY. 

In  the  centre  of  the  cylinder  is  a  column  which 
communicates  with  the  water  chamber  of  the 
cylinder,  and  from  it  pass  four  partitions,  which 
serve  to  support  the  test-tubes. 

In  the  lid  are  three  openings,  one  of  which  com- 
municates with  the  water  chamber  in  the  lid  by 
which  the  latter  is  filled,  and  into  which  a  thermo- 
meter is  then  fixed.  In  the  centre  an  opening 
admits  a  thermometer,  which  passes  into  the 
central  pipe  of  the  cylinder  ;  through  a  third  open- 
ing a  thermometer  passes  to  the  cavity  of  the 
cylinder.  The  cylinder  and  cover  are  jacketed 
with  felt,  and  the  apparatus  is  supported  on  iron 
legs. 

Serum  Inspissator.  —  A  shallow  tin  case  with 
glass  cover,  both  case  and  cover  jacketed  with  felt 

(Fig.  15).  The  case 
is  double  walled,  and 
the  water  contained 
in  the  interspace  is 
heated  from  below. 
It  is  supported  on 
four  legs,  and  the 

front  OneS   move 


FIG.  IS.-SERUM  INSPISSATOR. 

in     grooves    in    the 

case,  so  that  the  latter  can  be  placed  obliquely  at 
the  angle  required,  and  secured  in  position  by 
screw-clamps.  It  is  employed  for  coagulating 
sterile  liquid  serum,  and  for  solidifying  nutrient 
agar-agar  so  as  to  give  them  a  sloping  surface. 


APPARATUS,  MATERIAL,  AND  REAGENTS.    3! 

Glass  Capsules. — Small  capsules  or  hollowed- 
out  cubes  of  crystal  glass  are  employed  for  cultiva- 
tions on  solid  blood  serum,  on  nutrient  gelatine, 
and  on  agar-agar.  They  may  be  procured  of 
white  and  blackened  glass,  and  are  provided  with 
glass  slips  as  covers. 


(K)    APPARATUS    FOR    STORING,  AND    FOR   CULTI- 
VATIONS   IN,    LIQUID    MEDIA. 

Lister's  Flasks. — Professor  Lister  devised  a 
globe-shaped  flask  with  two  necks ;  a  vertical  and 
a  lateral  one.  The  lateral  one  is  a  bent  spout, 
tapering  towards  its  constricted  extremity.  When 
the  vessel  is  restored  to  the  erect  position  after 
pouring  out  some  of  its  contents,  a  drop  of  liquid 
remains  behind  in  the  end  of  the  nozzle,  and 
prevents  the  regurgitation  of  air  through  the  spout. 
A  cap  of  cotton  wool  is  tied  over  the  orifice,  and 
the  residue  in  the  flask  kept  for  future  use.  The 
vertical  neck  of  the  flask  is  plugged  with  sterilised 
cotton  wool  in  the  ordinary  way. 

Sternberg's  Bulbs. — Professor  Sternberg,  of 
America,  advocates  the  use  of  a  glass  bulb,  provided 
with  a  slender  neck  drawn  out  to  a  fine  point  and 
hermetically  sealed.* 

Aitken's  Test-tube. — This  is  an  ingenious 
device  for  counteracting  the  danger  of  entrance  of 

*  Magnin  and  Sternberg,  Bacteria.     1884. 


32  BACTERIOLOGY. 

atmospheric  germs  on  removal  from  the  ordinary 
test-tube  of  the  cotton  wool  plug.  Each  test-tube 
is  provided  with  a  lateral  arm  tapering  to  a  fine 
point,  which  is  hermetically  sealed. 

Drop-culture  Slides. — About  a  dozen  or  more 
thick  glass  slides  with  a  circular  excavation  in  the 
centre  are  required  for  drop-cultures. 

Vaseline. — A  small  pot  of  vaseline  with  a 
camel's  hair  brush  should  be  reserved  especially  for 
use  in  the  preparation  of  drop- cultures. 

Bulbed  Tubes. — Glass  vessels  such  as  test- 
tubes,  flasks,  and  pipettes,  which  are  used  in 
dealing  with  liquid  media,  have  already  been 
mentioned  under  other  headings,  but  bulbed  tubes, 
Pasteur's  bulbs,  and  various  other  forms  are  also 
required  for  special  experiments. 

(L)    APPARATUS    FOR  INCUBATION. 

There  are  several  forms  of  incubator,  each  of 
which  has  its  advocates.  They  are  mostly  rec- 
tangular chests,  with  glass  walls,  front  and  back, 
or  in  front  only.  A  cylindrical  model  is  preferred  by 
some.  Two  only  will  be  described  here,  D'Arsonval's 
and  Babes'.  The  former  admits  of  very  exact 
regulation  of  temperature,  and  the  latter  is  a  very 
practical  form  for  general  use. 

D'Arsonval's  Incubator.— The  "£tuve  HAr- 
sonval"  (Fig.  16)  is  a  very  efficient  apparatus, 
and  is  provided  with  a  heat  regulator,  which 


APPARATUS,    MATERIAL,    AND    REAGENTS.          33 

enables  the  temperature  to  be  maintained  with  a 
minimum  variation.  It  consists  of  a  cylindrical 
copper  vessel,  with  double  walls,  enclosing  a  wide 
interspace  for  containing  a  large  volume  of  water. 
The  roof  of  the 
water  -  chamber  is 
oblique,  so  that  the 
wall  rises  higher 
on  one  side  than 
on  the  other.  This 
admits  of  the  inter- 
space being  com- 
pletely  filled  with 
water.  At  the 
highest  point  is  an 
opening  fitted  with 
a  perforated  caout- 
chouc  stopper, 
through  which  a 
glass  tube  passes. 
The  mouth  of  the 
cylinder  itself  is 
horizontal,  and  is 

,          ,      ,  ,.,  FIG.  16.— D'ARSONVAL'S  INCUBATOR. 

closed    by    a    lid, 

which  is  also  double-walled  to  contain  water.  In 
the  lid  are  four  openings ;  one  serves  for  filling  its 
water-chamber,  and  the  others  for  thermometers  and 
for  regulating  the  air  supply  in  the  cavity  of  the  cylin- 
der. The  cylinder  is  continued  below  by  a  cone,  also 
double-walled,  and  there  is  a  perforated  grating  at 

3 


34 


BACTERIOLOGY. 


the  line  of  junction  of  the  cylinder  and  cone.  The 
cone  terminates  in  a  projecting  tube  provided  with 
an  adjustable  ventilator.  The  apparatus  is  fixed 
on  three  supports  united  to  one  another  below. 
One  of  them  is  utilised  for  adjusting  the  height  of 
the  heating  apparatus.  Situated  above  this  leg  is  the 
heat  regulating  apparatus  (Fig.  17),  attached  to  a 
circular,  lipped  aperture  in  the  outer  wall  of  the  in- 
cubator. To  the  lip  is 
fixed  with  six  screws  the 
corresponding  lip  of  a 
brass  box,  with  a  tightly 
stretched  diaphragm  of 
india-rubber  intervening. 
Thus  the  diaphragm 
FIG.  17.— SCHLOSING'S  MEMBRANE  separates  the  cavity  of 

REGULATOR.  ,        ,  r  , 

the   box   from   the   water 

in  the  interspace  of  the  incubator.  The  cap  of 
the  box,  which  screws  on,  is  bored  in  the  centre 
for  the  screw- pipe,  by  which  the  gas  is  supplied. 
Another  pipe  entering  the  box  from  below  is 
connected  with  the  gas  burners.  Around  the  end 
of  the  screw-pipe  a  collar  loosely  fits,  and  is  pressed 
against  the  diaphragm  by  means  of  a  spiral  wire 
spring.  Close  to  the  mouth  of  the  screw-pipe 
a  small  opening  exists,  so  that  the  gas  supply  to 
the  burners  is  not  entirely  cut  off  even  when  the 
diaphragm  completely  occludes  the  mouth  of  the 
screw-pipe. 

To  work  the   apparatus   the   tube   and    plug   must   be 


APPARATUS,    MATERIAL,    AND    REAGENTS.          35 


removed,  and  the  water-chamber  filled  completely  with 
distilled  or  rain  water  at  the  temperature  required.  The 
caoutchouc  plug  is  replaced  and  the  tube  placed  in  position. 
Gas  enters  through  d  (Fig.  17),  and  passes  through  the 
opening  at  its  extremity  into  the  chamber  of  the  box. 
Thence  it  passes  through  the  vertical  exit  which  is  connected 
with  the  gas  burners.  As  the  temperature  rises  the  water 
rises  in  the  tube,  and  at  the  same  time  exercises  a  pressure 
on  every  part  of  the  walls  of  the  incubator,  and  hence  on 
the  diaphragm.  In  consequence  of  this,  the  diaphragm 
bulging  outwards  approaches  the  end  of  the  tube  d,  and 
gradually  diminishes  the  gas  supply.  As  a  result  the 
temperature  falls,  the  water  contracts  and  sinks  in  the 
tube,  and  the  diaphragm  receding  from  d,  the  gas  supply 
is  again  increased.  By  adjusting  the  position  of  the  tube 
d  to  the  diaphragm,  any  required  temperature  within  the 
limits  of  the  working  of  the  apparatus  can  be  regulated  to 
the  tenth  of  a  degree ;  provided,  (i)  that  the  gas  supply  is 
rendered  independent  of  fluctua- 
tions of  pressure,  by  means  of  a 
gas-pressure  regulator,  (2)  that  the 
height  of  the  water  in  the  tube 
is  controlled  daily  by  the  with- 
drawal or  addition  of  a  few  drops 
of  distilled  water,  and  (3)  that 
the  apparatus  is  kept  in  a  place 
with  as  even  a  temperature  as  pos- 
sible, and  sheltered  from  currents 
of  air. 

The  burners  in  Fig.  1 6  are  pro- 
tected with  mica  cylinders  similar 
to  the  burner  represented  in  Fig.  18. 
The  flames  of  these  burners  can  be  turned  down  to 
the  smallest  length  without  danger  of  extinction,  and  the 
temperature  may  be  regulated  very  satisfactorily  without 
using  the  heat  regulator  just  described,  if  the  gas  first 


FIG.  18. 

GAS  BURNER  PROTECTED 
WITH  MICA  CYLINDER. 


BACTERIOLOGY. 


passes  through  a  pressure  regulator  (Fig.  19).     To  provide 

against  the  danger 
resulting  from  acci- 
dental extinction  of 
the  gas,  Professor 
Koch  has  devised  a 
/»  self-acting  apparatus 
(Fig.  20),  which, 
simultaneously  with 
the  extinction  of  the 
flame  of  the  burner, 
shuts  off  the  supply 
of  gas. 

Babes'  Incuba- 
tor.— The  pattern 
of  Dr.  Babes  is 


MOITESSIER'S  GAS-PRESSURE  REGULATOR. 


very  simple,  and    is    recommended   by  the  author 
in  preference  to   all  others   (Fig.    21). 


PIG.  20.— KOCH'S  SAFETY  BURNER. 

It  consists   of  a  double-walled  chest  with  sides 


APPARATUS,  MATERIAL,  AND  REAGENTS.    37 

and  roof  jacketed  with  felt.  Water  fills  the  inter- 
space between  the  walls, 
and  on  the  roof  are  two 
apertures,  one  for  a  gas 
regulator,  and  the  other 
for  a  thermometer.  In 
front,  the  chest  is  closed 
in  by  a  sheet  of  felt,  a 
glass  door,  and  a  sliding 
glass  panel.  The  appa- 
ratus can  be  suspended 
on  the  wall  or  supported 
on  legs,  and  is  heated 
from  below  by  means  of 
protected  burners. 
The  gas  should  pass 

FIG.  21.— BABES'  INCJUIJATGR. 

first  through  a  pressure- 
regulator,  and  then  through  a  thermo-regulator  to 
the  burners. 

Moitessier's  Gas-pressure  Regulator. — This 
apparatus  is  best  explained  by  reference  to  the 
diagram  (Fig.  19).  In  the  bottom  of  the  cylinder  A 
are  the  entrance  (k)  and  exit  (/)  gas  tubes.  The 
tap  (m)  regulates  the  size  of  the  flame.  The  cover 
(n  n)  roofs  in  the  cylinder  A.  The  bell  (B)  supports 
by  means  of  e  and/"  the  ball  valve  d,  which  lies  in  the 
cover  c  c.  The  gas,  entering  by  k,  passes  through 
the  valve  d,  and  is  thence  conducted  by  the  tube  a 
to  the  tube  /.  The  bell  B  and  the  weighted  dish 
h  are  screwed  on  to  the  connecting  red  g.  To 
diminish  as  much  as  possible  the  friction  of  g  in 


38  BACTERIOLOGY. 

z,  g  only  touches  /  by  three  projecting  ridges. 
Section  of  i  and  g  is  shown  at  s.  To  put  the  ap- 
paratus in  use  it  is  first  levelled,  then  h  is  screwed 
off  and  the  cover  n  n  removed.  A  mixture  of  two 
parts  of  pure  acid-free  glycerine  to  one  of  distilled 
water  is  poured  into  the  cylinder  until  it  flows  out  at 
q,  which  is  then  closed,  and  the  cover  n  n  replaced. 
The  manometers  are  filled 'with  coloured  water,  and 
k  and  /  connected  with  the  entrance  and  exit  gas 
tubing  respectively.  The  pressure  of  the  incoming 
gas  raises  the  bell  B  ;  and  with  it  the  valve  d  is 
raised  towards  the  opening  at  c  c.  The  weight  h, 
which  is  replaced  on  g,  by  its  downward  pressure 
counteracts  this  upward  pressure  of  the  gas  and 
opens  the  valve  c  c.  Thus  the  flame  is  best  regu- 
lated in  the  morning,  when  the  pressure  is  at  a 
minimum;  then  supposing  an  increase  of  pressure 
occurs,  the  weight  of  h  is  overbalanced,  B  is  raised 
and  with  it  d,  and  the  gas  supply  proportionately 
diminished  by  the  gradual  closing  of  the  valved 
opening. 

Reichert's  Thermo-Regulator. — This  regu- 
lator (Fig.  22)  consists  of  three  parts — a  hollow  T 
piece,  a  stem,  and  a  bulb.  The  T  piece  fits  like  a 
stopper  in  the  upper  widened  portion  of  the  stem. 
One  arm  of  the  T  is  open,  and  connected  with  the 
gas  supply ;  the  vertical  portion  terminates  in  a 
small  orifice,  and  is  also  provided  with  a  minute 
lateral  opening.  The  stem  is  provided  with  a 
lateral  arm,  and  this  arm,  the  stem,  and  the  bulb 


APPARATUS,    MATERIAL,    AND    REAGENTS.          39 

contain  mercury.  The  regulator  is  fixed  in  the 
roof  of  the  incubator,  so  that  the  bulb  projects 
either  into  the  interior  of  the  incubator  or  into 
the  water  chamber.  When  the  incu- 
bator reaches  the  required  temperature, 
the  mercury  is  forced  up  by  means  of 
the  screw  in  the  lateral  arm,  until  it 
closes  the  orifice,  at  the  extremity  of 
the  vertical  portion  of  the  T.  The  gas 
which  passes  through  the  lateral  orifice 
is  sufficient  to  maintain  the  apparatus 
at  the  required  temperature.  If  the 
temperature  of  the  incubator  falls  the 
mercury  contracts,  and  gas  passing  REGULATOR- 
through  the  terminal  orifice  of  the  T,  increases 
the  flame  of  the  burner  and  the  temperature  is 
restored. 

Page's  Thermo  -  Regulator  resembles  the 
above,  but  instead  of  the  T  piece  there  are  two 
pieces  of  glass  tubing.  The  outer  tubing  envelopes 
the  upper  part  of  the  stem  of  the  regulator,  and 
admits  of  being  raised  or  lowered.  The  upper  end 
of  this  tubing  is  closed  by  a  cork,  which  is  perforated 
to  admit  the  narrow  glass  tubing,  which  represents 
the  vertical  arm  of  the  T  passing  within  the  stem  of 
the  regulator.  This  has  a  terminal  and  a  lateral 
opening,  and  is  the  means  of  entrance  for  the  gas. 
This  regulator  is  adjusted  by  noting  when  the 
thermometer  indicates  the  desired  temperature,  and 
then  pushing  down  the  outer  tube  until  the  terminal 


40 


BACTERIOLOGY. 


opening-  of  the  inner  tube,  which  is  carried  down 
with  it,  is  obstructed  by  the  mercury. 

Meyer's  Thermo-Regulator  is  represented  in 
Fig.   23.      No.   I.    shows   the   construction    of   the 


FIG.  23.— MEYER'S  THERMO-REGULATOR. 

regulator  ;  its  inner  tube  terminates  in  an  oblique 
opening,  and  is  also  provided  with  a  minute 
lateral  aperture,  which  prevents  the  complete 
shutting  off  of  the  gas  supply.  No.  II.  illustrates 


APPARATUS,    MATERIAL,    AND    REAGENTS.          41 

the  method  of  introducing  the  mercury  by  suction 
through  a  filling  tube,  which  is  substituted  for  the 
inner  tube  of  the  regulator.  No.  III.  represents 
Frankel's  modification  of  the  same  instrument. 

'  (M)    INOCULATING  AND  DISSECTING  INSTRUMENTS  AND 
APPARATUS  IN    COMMON    USE. 

Mouse  Cages. — As  mice  are  the  animals  most 
frequently  employed  for  experimental  purposes, 
mouse  cages  have  been  especially  introduced,  con- 
sisting simply  of  a  cylindrical  glass  jar  with  a 
weighted  wire  cover. 

Dressing-case. — A  small  surgical  dressing- 
case,  with  its  usual  accessories — forceps,  knives, 
small  straight  and  curved  scissors,  needles,  silk, 
and  so  forth — will  serve  for  most  purposes. 

Pravaz'  Syringe. — Koch's  modification  of 
Pravaz'  syringe  admits  of  sterilisation  by  exposure 
to  1 50°  C.  for  a  couple  of  hours. 

Special  Instruments  and  Material. — Instru- 
ments required  for  special  operations  and  the 
materials  necessary  for  strict  antiseptic  precautions 
need  not  be  detailed  here.* 

Dissecting  Boards. — Slabs  of  wood  in  various 
sizes,  or  gutta  percha  trays,  provided  with  large- 
headed  pins,  are  employed  for  ordinary  purposes. 

Dissecting  Case. — A  dissecting  case  fitted  with 
scalpels,  scissors,  hooks,  etc.,  should  be  reserved 
entirely  for  post-mortem  examinations. 

*  Vide  Cheyne,  Antiseptic  Stirgery.     1882. 


42 


BACTERIOLOGY. 


(N)    GENERAL    LABORATORY    REQUISITES. 

Siphon  Apparatus. — Two  half-gallon  or  gallon 
glass  bottles,  with  siphons  connected  with  long 
flexible  tubes  provided  with  glass  nozzles  and 
pinchcocks  (Fig.  24),  should  be  employed  for  the 


FIG.  24. 

SIPHON  BOTTLE,  WITH  FLEXIBLE  TUBE,  GLASS  NOZZLE,  AND 
A  MOHR'S  PINCHCOCK. 


following  purposes  : — One  is  used  to  contain  distilled 
water,  with  the  nozzle  hanging  down  conveniently 
within  reach  of  the  working  table ;  the  other  is  to 
contain  a  solution  of  corrosive  sublimate  (i  in  1000), 
and  may  be  placed  so  that  the  nozzle  hangs  close 
to  the  lavatory  sink  or  basin.  The  former  replaces 
the  use  of  the  ordinary  wash  bottle,  in  washing  off 


APPARATUS,    MATERIAL,    AND    REAGENTS.          43 

surplus  stain  from  cover  glasses,  etc.,  and  the  latter 
is  conveniently  placed  for  disinfection  of  vessels  and 
hands  after  cleansing  with  water.  They  should  be 
placed  on  the  top  of  a  cupboard,  or  on  a  high 
shelf. 

Desiccator — The  Desiccator  (Fig.  25)  consists 
of  a  porcelain  pan  containing  concentrated  sulphuric 
acid,  and  covered  over  with  a  bell-glass  receiver. 


FIG.  25. — DESICCATOR. 

The  sheet  of  plate-glass  upon  which  the  pan  rests 
is  ground  upon  its  upper  surface,  and  the  rim  of 
the  glass  bell  is  also  ground  and  well  greased.  In 
the  centre  of  the  pan  is  a  column  supporting  a 
circular  frame,  which  is  covered  with  wire  gauze. 
Slices  of  potatoes,  upon  which  micro-organisms 
have  been  cultivated,  are  rapidly  dried  by  the 
action  of  the  sulphuric  acid  in  confined  air.  A 
cultivation  of  Bacterium  prodigiosum,  for  example, 


44  BACTERIOLOGY. 

may   be   dried    in    this   way,    and    preserved    for 
subsequent  experiments. 

Other  items  commonly  in  use  in  a  research 
laboratory  cannot  be  detailed  here,  and  a  descrip- 
tion of  air-pumps,  refrigerators,  etc.,  access  to 
which  is  nevertheless  necessary  for  some  special 
investigations,  must  be  sought  for  elsewhere.* 

*  All  bacteriological  apparatus,  as  employed  by  Professor  Koch, 
may  be  obtained  from  Dr.  Muencke,  58,  Louisen  Strasse,  Berlin. 
Nearly  all  the  figures  of  apparatus  here  given  are  from  blocks,  kindly 
lent  to  me  by  Dr.  Muencke.  Griffin  6X  Son,  22,  Garrick  Street, 
Covent  Garden,  W.C.,  will  make  to  order  any  bacteriological  ap- 
paratus required,  and  from  them  all  glass  vessels  and  chemical 
apparatus  of  home  manufacture  can  be  obtained.  All  histological 
instruments  and  material,  such  as  microscopes,  microtomes,  aniline 
dyes,  celloidin,  gelatine,  agar-agar,  etc.,  are  supplied  by  G.  Konig, 
Berlin,  N.W.,  35,  Dorotheen  Strasse.  Chemicals,  staining  reagents, 
and  ready- prepared  nutrient  gelatine  can  also  be  obtained  from  Dr. 
Georg  Griibler,  Leipsig,  17,  Dufour  Strasse.  Solutions  of  lithium- 
carmine,  picro-lithium  carmine  (Orth.),  picro-carmine  (Weigert), 
alum  and  borax-carmine  (Grenacher),  etc.,  ready  for  use,  are  pre- 
pared by  Becker  &  Co.,  34,  Maiden  Lane,  Covent  Garden,  London, 
W.C.  The  latter  firm  also  keep  in  stock  bacteriological  apparatus 
and  glass  ware  of  the  German  pattern. 

Mr.  Baker,  of  High  Holborn,  W.C.,  is  recommended  for  the 
supply  of  microscopes  and  the  ordinary  objectives  by  continental 
makers,  and  the  new  apochromatic  objectives  recently  introduced 
by  Zeiss.  Objectives  made  of  the  new  glass  are  also  constructed  by 
Powell  and  Leland,  but  though  invaluable  to  the  specialist  their 
expense  places  them  beyond  the  reach  of  the  general  student. 
Messrs.  Swift  &  Son  have  recently  introduced  an  excellent  -fa  oil. 
imm.  for  five  guineas,  and  are  prepared  to  supply  a  microscope 
completely  equipped  for  bacteriological  work  at  a  very  low  price. 


CHAPTER  III. 

MICROSCOPICAL  EXAMINATION  OF  BACTERIA  IN 
LIQUIDS,  IN  CULTIVATIONS  ON  SOLID  MEDIA, 
AND  IN  TISSUES. 

Preliminary  Remarks.  —  In  conducting-  bac- 
teriological researches,  the  importance  of  absolute 
cleanliness  cannot  be  too  strongly  insisted  upon. 
All  instruments,  glass  vessels,  slides,  and  cover 
glasses  should  be  thoroughly  cleansed  before 
use.  A  wide-mouthed  glass  jar  should  always 
be  close  at  hand,  containing  refuse  alcohol  for 
the  reception  of  rejected  slide  preparations,  or 
dirty  cover-glasses.  When  required  again  for 
use,  slides  can  be  easily  wiped  clean  with  a  soft 
rag.  Cover-glasses  require  further  treatment,  for 
unless  they  are  perfectly  clean  it  is  difficult  to 
avoid  the  presence  of  air  bubbles  when  mounting 
specimens.  They  should  be  left  in  strong  acid 
(hydrochloric,  sulphuric,  or  nitric)  for  some  hours ; 
they  are  then  washed,  first  with  water  and  then 
with  alcohol,  and  carefully  wiped  with  a  soft  rag. 
The  same  principle  applies  in  the  preparation 
and  employments  of  culture  media  ;  any  laxity  in 
the  processes  of  sterilisation,  or  insufficient  atten- 


46  BACTERIOLOGY. 

tion  to  minute  technical  details,  will  surely  be 
followed  with  disappointing  results  in  the  contami- 
nation of  one's  cultures,  resulting  in  the  loss  of 
much  time.  When  using  platinum  needles,  either 
for  inoculating  fresh  tubes  in  carrying  on  a  series 
of  pure  cultures,  or  in  transferring  a  small  portion 
of  a  cultivation  to  a  cover-glass  for  examination 
under  the  microscope,  the  careful  sterilisation  of 
the  needle  by  heating  the  platinum  wire  till  it  is 
white  hot  in  every  part,  and  heating  also  as  much 
of  the  glass  rod  as  is  made  to  enter  the  test- 
tube,  must  be  carried  out  with  scrupulous  care. 
Indeed  it  is  a  good  plan  to  let  it  become  a 
force  of  habit  to  sterilise  the  needle  before  and 
after  use  on  every  occasion,  whatever  may  be  the 
purposes  for  which  it  is  employed. 

(A)    EXAMINATION    IN    THE    FRESH    STATE. 

Liquids  containing  micro-organisms  such  as 
pus,  blood,  juices,  culture-fluids,  can  be  investigated 
by  transferring  a  drop  with  a  sterilised  ose  or  a 
capillary  pipette  to  a  slide,  covering  it  with  a 
clean  cover-glass,  and  examining  without  further 
treatment.  If  it  is  desirable  to  keep  the  specimen 
under  prolonged  observation,  a  drop  of  sterilised 
water  or  salt  solution  must  be  run  in  at  the 
margin  of  the  cover-glass  to  counteract  the 
tendency  to  dry.  Cultures  on  solid  media  can  be 
examined  by  transferring  a  small  portion  with  a 


MICROSCOPICAL    EXAMINATION    OF   BACTERIA.     47 

sterilised  needle  to  a  drop  of  sterilised  water  on 
a  slide,  thinning  it  out,  and  covering  with  a 
cover-glass  as  already  described.  A  more  satis- 
factory method,  by  which  one  can  keep  micro- 
organisms under  observation  and  study  their 
movements,  spore-formation,  etc.,  will  be  described 
under  " Drop-cultures. "  Tissues  in  the  fresh  state 
may  be  teased  out  with  needles  in  sterilised  salt 
solution,  and  pressed  out  into  a  sufficiently  thin 
layer  between  the  slide  and  cover-glass.  Glycerine 
may  in  many  cases  be  substituted  for  salt  solution, 
especially  for  the  examination  of  micro-organisms 
such  as  Actinomyces,  Aspergilli,  etc. 

There  is  as  a  rule  no  difficulty  in  recognising 
the  larger  micro-organisms  such  as  those  just 
mentioned,  but  where  we  have  to  deal  with  very 
small  bacilli,  bacteria  and  micrococci,  they  may 
possibly  be  mistaken  for  granular  detritus  or  fat- 
crystals,  or  vice-versa.  They  are  distinguished  by 
the  fact  that  fatty  and  albuminous  granules  are 
altered  or  dispersed  by  acetic  acid,  and  changed  by 
solution  of  potash ;  alcohol,  chloroform,  and  ether 
dissolve  out  fat  crystals  or  fatty  particles ;  on  the 
other  hand,  micro-organisms  remain  unaffected  by 
these  re-agents.  This  micro-chemical  reaction  is 
made  the  basis  of  Baumgarten's  method  (p.  278). 


48  BACTERIOLOGY. 

(B)    COVER-GLASS-PREPARATIONS. 

The  method  next  to  be  described  is  the  most 
commonly  employed;  in  addition  to  its  value  as 
a  means  of  examining  liquids,  etc.,  it  affords  the 
additional  advantage  of  enabling  one  to  make, 
if  necessary,  a  large  number  of  preparations  which 
when  dried  can  be  preserved,  stained  or  unstained, 
in  ordinary  cover-glass  boxes ;  they  are  then  in 
a  convenient  form  for  transport,  and  can  be 
mounted  permanently  at  leisure. 

The  method  is  as  follows : — A  cover-glass  is 
smeared  with  the  cut  surface  of  an  organ,  or 
pathological  growth,  or  with  sputum  ;  or  a  drop 
of  blood,  pus,  or  other  fluid  to  be  examined,  is 
conveyed  to  it  with  a  large  ose.  By  means  of 
another  cover-glass,  the  juice,  or  fluid,  is  squeezed 
out  between  them  into  a  thin  layer,  and  on  sliding 
them  apart  each  cover-glass  bears  on  one  side  a 
thin  film  of  the  material  to  be  examined.  They 
are  then  placed  with  the  prepared  side  upwards  and 
allowed  to  dry.  After  a  few  minutes,  they  are 
held  with  a  pair  of  flat-bladed  or  spring  forceps, 
with  the  prepared  side  uppermost,  and  passed 
rapidly  three  times  through  the  flame  of  a  spirit 
lamp  or  Bunsen  burner.  To  stain  them,  put  two 
or  three  drops  of  an  aqueous  solution  of  fuchsine 
or  methyl  violet  over  the  film,  and  after  a  minute 
or  two  wash  off  the  surplus  stain  with  distilled 
water  by  means  of  the  siphon  apparatus  or  a  wash 


MICROSCOPICAL   EXAMINATION    OF   BACTERIA.    49 

bottle.  Turn  the  cover-glass  on  to  a  slide,  remove 
excess  of  water  with  filter  paper,  and  wipe  the 
exposed  surface ;  examine  with  Zeiss'  DD  (about 
230  diams.),  and  if  a  higher  power  be  required, 
which  is  usually  the  case,  place  a  droplet  of  cedar 
oil  on  the  cover  glass,  and  examine  with  an  im- 
mersion lens. 

If  the  specimen  is  to  be  made  permanent,  fix  the 
cover-glass  at  one  corner  with  the  thumb,  and  with 
a  soft  rag  carefully  wipe  off  the  cedar  oil ;  then 
float  off  the  cover-glass  by  running  in  distilled 
water  at  its  margin,  and  having  made  a  little  ledge 
with  a  strip  of  filter-paper,  place  the  cover-glass  up 
against  it  upon  one  of  its  edges  and  leave  it  to  dry. 
When  perfectly  dry  mount  in  Canada  balsam,  or 
put  it  away  in  a  cover-glass  box  provided  with  a 
label  of  contents. 

A  culture  from  a  solid  medium  may  be  stained 
and  examined  in  the  same  way  after  spreading  it 
out  with  a  needle  into  a  thin  film,  with  or  without 
the  addition  of  a  droplet  of  sterilised  water. 

In  many  cases  it  is  necessary  or  preferable  to 
apply  the  stain  for  a  much  longer  period.  This  is 
effected  by  pouring  some  of  the  staining  solution 
into  a  watch-glass,  and  allowing  the  cover-glasses 
to  swim  on  the  surface,  with  their  prepared  side,  of 
course,  downwards.  Throughout  all  these  manipu- 
lations it  is  necessary  to  bear  in  mind  which  is  the 
prepared  surface  of  the  cover-glass. 

Double  coloration  of  cover-glass  prepara- 


5<D  BACTERIOLOGY. 

tions  can  also  be  obtained  as  in  Ehrlich's  method 
for  staining  tubercular  sputum,  or  by  staining  with 
eosin  after  treatment  by  the  method  of  Gram. 

Ehrlich's  Method  is  as  follows  : — Five  parts  of 
aniline  oil  are  shaken  up  with  one  hundred  parts  of 
distilled  water,  and  the  emulsion  filtered  through 
moistened  filter  paper.  A  saturated  alcoholic  solu- 
tion of  fuchsine,  methyl  violet,  or  gentian  violet,  is 
added  to  the  filtrate  in  a  watch-glass  drop  by  drop 
until  precipitation  commences.  Cover-glass  pre- 
parations are  floated  in  this  mixture  for  fifteen 
minutes  to  half  an  hour,  then  washed  for  a  few 
seconds  in  diluted  nitric  acid  (one  part  nitric  acid  to 
two  of  water)  and  then  rinsed  in  distilled  water. 
The  stain  is  removed  from  everything  except  the 
bacilli,  but  the  ground  substance  can  be  after- 
stained,  brown  if  the  bacilli  are  violet,  or  blue  if 
they  have  been  stained  red  (Plate  XX.,  Fig.  i). 

Double  staining  with  eosin  after  the  method  of 
Gram  is  described  under  tissue  staining.  The 
cover-glass  preparations  are  treated  by  the  same 
processes  as  employed  with  sections  ;  superfluous  oil 
of  cloves  can  be  removed  by  gently  pressing  the 
cover-glass  between  double  layers  of  filter  paper. 

Babes'  Method  affords  a  very  rapid  means  of 
examining  cultivations,  etc.  -A  little  of  the  growth, 
removed  by  means  of  a  sterilised  platinum  hook  or 
small  ose,  is  spread  out  on  a  cover  glass  into  as  thin 
a  film  as  possible :  when  almost  dry,  a  drop  or  two 
of  a  weak  aqueous  solution  of  methyl  violet  is 


MICROSCOPICAL   EXAMINATION    OF   BACTERIA.     51 

allowed  to  fall  from  a  pipette  upon  the  film.  The 
cover-glass  with  the  drop  of  stain  is  after  a  minute 
carefully  turned  over  on  to  a  slide,  and  the  excess 
of  stain  gently  and  gradually  removed  by  pressure 
with  a  strip  of  filter  paper.  It  affords  a  rapid 
means  of  demonstration,  for  example  of  such  a 
cultivation  as  Koch's  comma  bacilli  in  nutrient 
gelatine,  enabling  the  microbes  to  be  seen  in  some 
parts  of  the  preparation  both  stained  and  in  active 
movement. 

His*  Method. — The  staining  of  fresh  prepara- 
tions, especially  those  with  no  coagulable  albumen 
to  fix  them,  may  be  also  carried  out  by  His'  method. 
A  slide  is  prepared  as  already  described  in  the 
examination  of  micro-organisms  in  the  fresh  state. 
The  reagents  are  then  applied  by  placing  them 
with  a  pipette  drop  by  drop  at  one  margin  of 
the  cover-glass,  and  causing  them  to  flow  through 
the  preparation  by  means  of  a  strip  of  filter  paper 
placed  at  the  opposite  margin. 

To  stain  spores  the  method  described  on  p.  48 
is  somewhat  modified.  The  cover-glass  prepara- 
tions may  be  either  passed  as  many  as  twelve  times 
through  the  flame,  or  heated  to  a  temperature  of. 
210°  for  half-an-hour,  or  exposed  to  the  action  of 
strong  sulphuric  acid  for  a  few  seconds,  and  then 
stained  with  a  watery  solution  of  the  dye. 

To  double-stain  spore-bearing  bacilli. — The 
cover-glass  preparations  may  be  floated  for  twenty 
minutes  on  a  fuchsine  aniline-water  solution,  as 


5  2  BACTERIOLOGY. 

used  in  Ehrlich's  method,  which  has  been  heated 
to  boiling-point.  The  fuchsine  is  removed  from 
the  bacilli  either  by  simply  rinsing  in  water,  in 
alcohol,  or  in  weak  acid,  according  to  the  species, 
and  then  the  preparations  are  floated  for  a  few 
minutes  on  solution  of  methylene  blue,  rinsed  in 
water,  dried  and  mounted. 

To  stain  flagella. — Koch  recommends  floating 
the  cover-glasses  on  a  concentrated  watery  solution 
of  haematoxylin.  From  this  they  are  transferred 
to  a  5  per  cent,  solution  of  chromic  acid  or 
to  Miiller's  fluid,  by  which  the  flagella  obtain 
a  brownish-black  coloration.  The  author  has 
succeeded  in  demonstrating  and  photographing 
flagella,  by  staining  with  a  drop  of  a  saturated 
solution  of  gentian  violet  in  absolute  alcohol. 
Before  the  alcohol  has  time  to  evaporate  the  cover- 
glass  is  rinsed  in  water,  and  then  allowed  to  dry, 
and  finally  mounted  in  balsam.  A  very  intense 
staining  of  the  whole  preparation  results. 

(C)    COVER-GLASS    IMPRESSIONS. 

One  of  the  most  instructive  methods  for  examin- 
ing micro-organisms  is  to  make  what  is  called  in 
German  a  " Klatsch  Praparat"  It  enables  us  in 
many  cases  to  study  the  relative  position  of  in- 
dividual micro-organisms  one  to  another  in  their 
growth  on  solid  cultivating  media,  and  in  some 
cases  produces  the  most  exquisite  preparations  for 


MICROSCOPICAL    EXAMINATION    OF    BACTERIA.     53 

the  microscope.  A  perfectly  clean,  usually  small- 
sized,  cover- glass  is  carefully  deposited  on  a  plate 
or  potato  culture,  and  gently  and  evenly  pressed 
down.  One  edge  is  then  levered  up,  carefully,  with 
a  needle  and  the  cover-glass  lifted  off  by  means  of 
forceps.  It  is  then  allowed  to  dry,  passed  through 
the  flame  three  times,  and  stained  as  already  de- 
scribed. In  the  case  of  plate-cultures,  especially 
where  no  liquefaction  has  taken  place,  the  growth 
is  bodily  transferred  to  the  cover-glass  and  a  vacant 
area  left  on  the  gelatine  or  agar-agar,  correspond- 
ing exactly  with  the  form  and  size  of  the  cover-glass 
employed  (Plate  XXV.,  Figs,  i  and  2). 


CHAPTER    IV. 

PREPARATION  AND   STAINING   OF  TISSUE 
SECTIONS. 

(A)    METHODS    OF    HARDENING    AND    DECALCIFYING 
PREPARATIONS. 

To  harden  small  organs,  such  as  the  viscera  of  a 
mouse,  they  must  be  placed  on  a  piece  of  filter 
paper  at  the  bottom  of  a  small,  wide-mouthed  glass 
jar,  and  covered  with  about  twenty  times  their 
volume  of  absolute  alcohol.  Larger  organs,  patho- 
logical growths,  etc.,  are  treated  in  the  same  way, 
but  must  first  be  cut  into  small  pieces,  or  cubes, 
varying  from  a  quarter  of  an  inch  to  an  inch  in 
size.  Muller's  fluid  may  also  be  employed,  and 
methylated  spirit  may  be  substituted  for  alcohol, 
from  motives  of  economy.  Tissues  hardened  in 
absolute  alcohol  are  ready  for  cutting  in  two  or 
three  days,  and  those  hardened  in  Muller's  fluid  in 
as  many  weeks. 

Teeth,  or  osseous  structures,  must  first  be  placed 
in  a  decalcifying  solution,  such  as  Kleinenberg's. 
When  sufficiently  softened  they  are  allowed  to  soak 


PREPARATION  AND  STAINING  OF  TISSUE   SECTIONS.  55 

in  water,  to  wash  out  the  picric  acid,  and  then 
transferred  through  weak  spirit  to  absolute  alcohol. 
Ebner's  solution  also  gives  excellent  results,  es- 
pecially when  the  structures  to  be  decalcified  are 
placed  in  fresh  solution  from  time  to  time. 


(B)     METHODS    OF     EMBEDDING,     FIXING,    AND 
CUTTING. 

Material  to  be  cut  with  the  freezing  microtome, 
if  hardened  in  spirit,  must  be  well  soaked  in  water 
before  being  frozen ;  if  hardened  in  Muller's  fluid, 
it  can  be  frozen  at  once. 

If  Williams'  microtome  is  employed,  the  hard- 
ened tissues  must  first  be  well  soaked  in  gum 
mucilage,  then  frozen,  and  cut. 

For  cutting  with  Jung's  microtome,  the  tissues 
are  embedded  in  paraffin,  or  celloidin,  and  mounted 
on  cork,  or,  if  firm  enough,  they  may  be  fixed 
upon  cork  without  any  embedding  material  at  all. 
Paraffin,  dissolved  in  chloroform,  will  be  found 
very  serviceable  as  an  embedding  material,  but 
celloidin  is  more  commonly  employed  now.  The 
pieces  of  tissue  to  be  embedded  are  placed,  after 
the  process  of  hardening  is  completed,  in  a  mix- 
ture of  ether  and  alcohol  for  an  hour  or  more. 
They  are  then  transferred  to  a  solution  of  celloidin 
in  equal  parts  of  ether  and  alcohol,  and  left  there, 
usually,  for  several  hours.  Meanwhile,  corks  ready 
cut  for  the  clamp  of  the  microtome  are  smeared 


56  BACTERIOLOGY. 

over  with  the  solution  of  celloidin  ;  this  is  applied 
with  a  glass  rod  to  the  surface  which  is  to  receive 
the  piece  of  tissue.  The  corks  are  then  set  aside 
for  the  film  of  eelloidin  to  harden.  The  pieces  of 
tissue  are  allowed  to  remain  in  the  celloidin  solution 
for  from  one  to  twenty-four  hours,  the  time  varying 
according  to  the  structure  of  the  specimen.  Better* 
results  are  obtained  in  the  case  of  lung,  or  de 
generated  broken-down  tissue,  if  left  for  a  much 
longer  time  than  is  found  to  be  sufficient  for  firmer 
structures.  The  specimen,  when  ready,  is  removed 
from  the  celloidin  solution  with  forceps,  and  placed 
upon  a  prepared  cork.  A  little  of  the  solution, 
which  is  of  syrupy  consistence,  is  allowed  to  fall 
on  the  piece  of  tissue  to  cover  it  completely,  and 
the  mounted  specimen  is  finally  placed  in  60  to  So 
per  cent,  alcohol  to  harden  the  celloidin.  The 
specimen  will  be  ready  for  cutting  next  day. 

The  specimen  may  be  more  neatly  embedded  by 
fixing  it  with  a  pin  in  a  small  paper  tray,  pouring 
the  celloidin  solution  over  it,  and  then  placing  the 
tray  in  alcohol  to  harden  the  celloidin.  The  em- 
bedded specimen  is  then  fixed  on  a  cork,  which 
has  been  cut  for  the  clamp  of  the  microtome.  The 
celloidin  in  the  section  disappears  in  the  process  of 
clearing  with  clove-oil. 

Material  infiltrated  with  paraffin  must  be  cut  per- 
fectly dry,  and  the  sections  prevented  from  rolling 
UP  by  gentle  manipulation  with  a  camePs-hair 
brush.  They  must  then  be  picked  off  the  blade  of 


PREPARATION  AND  STAINING  OF  TISSUE  SECTIONS.   57 

the  knife  with  a  clean  needle,  and  dropped  into  a 
watch-glass  containing1  xylol.  This  dissolves  out 
the  paraffin ;  the  sections  are  then  transferred  to 
alcohol  to  get  rid  of  the  xylol,  and  then  to  the 
staining  solution. 

In  the  case  of  specimens  embedded  in  celloidin, 
or  mounted  directly  on  a  cork,  the  tissue,  as  well  as 
the  blade  of  the  knife,  should  be  kept  constantly 
bathed  with  alcohol,  and  the  sections  transferred 
from  the  blade  with  a  camel's  hair  brush,  and 
floated  in  alcohol. 

For  fixing  small  organs  and  pieces  of  firm  tissue 
directly  on  cork,  such  as  the  kidneys  of  a  mouse, 
or  liver,  one  employs  gelatine,  or  glycerine- gelatine, 
liquefied  over  a  Bunsen  burner  in  a  porcelain  cap- 
sule. The  cork  with  specimen  affixed  is  placed  in 
alcohol,  and  is  ready  for  cutting  sections  next  day. 

The  advantage  of  glycerine-gelatine  consists  in 
that  it  may  be  used  for  fixing  irregular  pieces  of 
tissue,  as  it  does  not  become  of  a  consistency  that 
would  injure  the  edge  of  the  knife. 

(c)  GENERAL  PRINCIPLES  OF  STAINING  BACTERIA 
IN  TISSUE  SECTIONS:  METHODS  OF  WEIGERT, 
GRAM,  AND  WEIGERT-EHRLICH. 

Sections  of  fresh  tissues  made  with  the  freezing 
microtome  are  to  be  floated  and  well  spread  out 
in  *8  per  cent,  salt  solution,  and  then  carefully 
transferred,  well  spread  out  on  the  copper  lifter,  to 
a  watch-glass  containing  absolute  alcohol.  Simi- 


58  BACTERIOLOGY. 

larly  sections  selected  from  those  cut  with  Jung's 
microtome,  may  be  transferred  from  the  spirit  to 
absolute  alcohol.  The  sections  may  be  then  stained 
by  any  of  the  methods  to  be  described. 

It  is  often  advisable  to  employ  some  method 
which  will  enable  one  to  study  the  structure  of  the 
tissue  itself.  In  the  same  way  with  sections  how- 
ever prepared,  one  should  always  examine  with  a 
low  power  (Zeiss'  AA)  first;  this  enables  one  to 
recognise  the  tissue  under  examination  in  most 
cases,  and  even  to  examine  in  many  cases  the  topo- 
graphical distribution  of  masses  of  bacteria.  With 
Zeiss'  DD.,  Oc.  2,  a  power  of  about  250  diams., 
very  many  bacteria  can  be  distinguished,  and  with 
the  oil  immersion  lenses  the  minutest  bacilli  and 
micrococci  can  be  recognised,  and  the  exact  form 
of  individual  bacteria  accurately  determined.  As 
Zeiss'  microscopes  are,  like  most  good  modern 
instruments,  provided  with  a  triple  nosepiece,  there 
is  no  loss  of  time  in  examining  a  preparation  suc- 
cessively with  these  different  powers. 

Weigert's  Method. — A  very  useful  method  for 
staining  both  the  tissue  and  the  bacteria  is  as 
follows  : — Place  the  sections  for  from  six  to  eighteen 
hours  in  a  one  per  cent,  watery  solution  of  any  of 
the  basic-aniline  dyes  (methyl  violet,  gentian  violet, 
fuchsine,  bismarck  brown).  To  hasten  the  process 
place  the  capsule  containing  the  solution  in  the 
incubator,  or  heat  it  to  45°  C.  A  stronger  solution 
may  also  be  employed,  in  which  case  the  sections 


PREPARATION  AND  STAINING  OF  TISSUE  SECTIONS,  59 

are  far  more  rapidly  stained,  and  are  easily  over- 
stained.  In  the  latter  case  they  must  be  treated 
with  a  half- saturated  solution  of  carbonate  of 
potash.  In  either  case  the  sections  are  next  washed 
with  distilled  water,  and  passed  through  60  per  cent, 
alcohol  into  absolute  alcohol.  When  almost  de- 
colorised spread  out  the  section  carefully  on  a 
copper  lifter  and  transfer  it  to  clove-oil,  or  stain 
with  picro-carmine  solution  (Weigert's)  for  half-an- 
hour,  wash  in  water,  alcohol,  and  then  treat  with 
clove-oil.  After  the  final  treatment  with  clove- oil, 
transfer  with  the  copper  lifter  to  a  clean  glass  slide. 
Dry  the  preparation  by  pressure  with  a  piece  of 
filter  paper  folded  four  times,  and  preserve  in  Canada 
balsam  dissolved  in  xylol. 

Gram's  Method. — In  the  method  of  Gram  the 
sections  are  stained  for  three  minutes  in  aniline- 
gentian-violet  solution.  This  is  prepared  by  shak- 
ing up  one  ccm.  of  pure  aniline  with  twenty  four 
parts  of  water,  and  filtering  the  emulsion.  Haifa 
gramme  of  the  best  finely  powdered  gentian  violet 
is  dissolved  in  the  clear  filtrate,  and  the  solution 
filtered  before  use.  The  sections  are  then  trans- 
ferred to  a  solution  of  iodine  in  iodide  of  potassium 
till  they  become  dark  brown  in  colour,  and  then 
decolorised  in  absolute  alcohol.  The  time  required 
for  complete  decolorisation  in  alcohol  varies  from 
a  few  minutes  to  twenty-four  hours.  They  are 
then  treated  with  clove-oil  and  mounted  in  Canada 
balsam.  It  is  much  better,  however,  to  employ 


6O  BACTERIOLOGY. 

the    aniline-gentian-violet    solution    quite    freshly 
prepared,    and    the    following    useful    method    is 
invariably  used  by  the  author : — Place  four  or  five 
drops  of  pure  aniline  in  a  test-tube,  fill  it  three- 
quarters  full  with  distilled  water,  close  the  mouth 
of    the    tube    with    the    thumb   and   shake   it   up 
thoroughly.     Filter  the  emulsion  twice,  and  pour 
the  filtrate  into  a  watch-glass  or  glass  capsule.     To 
the  perfectly  clear  aniline  water  thus  obtained  add 
drop  by  drop  a  concentrated  alcoholic  solution  of 
gentian-violet  till  precipitation  commences.     Stain 
sections  in  this  solution  from  ten  minutes  to  half 
an    hour,    then    transfer    to   iodine-potassic-iodide 
solution,  and  decolorise  in  alcohol.     The   process 
of  decolorisation  may  be  hastened  by  placing   the 
section  in  clove-oil  and  returning  it  to  alcohol,  and 
again  to  clove-oil.     If  examined,  after  it  has  been 
finally    treated    with    clove-oil    and    mounted    in 
Canada  balsam,  the   tissue  appears   colourless   or 
tinged  faintly  yellow,  while  micro-organisms,  e.g., 
bacilli  and   micrococci,  are  stained  blue  or   blue- 
black.     Double  staining  is  obtained  by  transferring 
the    sections   after  decolorisation   to  a  solution  of 
eosin,  bismarck  brown,  or  vesuvin,  again   rinsing 
in  alcohol,  clearing  in  clove-oil,  and  mounting  in 
balsam.      Another  instructive  method  is  to  place 
the    decolorised     sections    in    picro-carminate    of 
ammonia  for  three  or  four  minutes,  and  then  treat 
with  alcohol,  oil  of  cloves,  and  balsam.     In  this  way 
the  nuclei  are  well  stained.     A  somewhat  similar 


PREPARATION  AND  STAINING  OF  TISSUE   SECTIONS.  6 1 

result  is  obtained  by  placing  the  sections  for  a  few 
minutes  in  Orth's  solution  (picro-lithium-carmine) ; 
transferring  to  acidulated  alcohol,  then  to  an  alco- 
holic solution  of  picric  acid,  and  treating  with  clove- 
oil  and  balsam  (Plate  XVII.,  Fig.  i). 

Weigert-Ehrlich  Method. — This  is  a  method 
in  which  nitric  acid  is  employed  as  a  decolorising 
agent.  It  is  as  follows : — Filtered,  saturated,  watery 
solution  of  aniline  one  hundred  parts ;  saturated 
alcoholic  solution  of  a  basic  aniline  dye  (methyl- 
violet,  gentian-violet,  fuchsine),  eleven  parts;  are 
mixed  and  filtered.  Rapid  staining  is  obtained  by 
warming  the  solution.  Decolorise  with  nitric  acid 
(i  in  2),  and  stain  with  a  contrast  colour.  As 
delicate  sections  are  apt  to  be  injured  by  immersion 
in  the  nitric  acid,  they  may  be  transferred  from  the 
fuchsine  solution  to  distilled  water,  then  rinsed  a 
few  minutes  with  alcohol,  and  finally  placed  in  the 
following  contrast  stain  for  one  to  two  hours  : — 

Distilled  water  .         .         .100  cc. 

Saturated  alcoholic  solution  of 

methylene  blue.        Y"        .         20  cc. 

Formic  acid  10  mm.* 

The  method  of  Weigert-Ehrlich  is  employed  for 
staining  both  leprosy  and  tubercle  bacilli.  The  ani- 
line-fuchsine  solution  may  also  be  prepared  by  the 
simple  plan  described  for  Gram's  method  (page  60). 
The  more  special  methods  for  staining  will  be  given 
with  the  description  of  those  species  of  micro- 
organisms to  which  they  apply. 

*  Watson  Cheyne,  Practitioner.     1883. 

•****' 


CHAPTER  V. 

PREPARATION  OF  NUTRIENT  MEDIA    AND 
METHODS  OF  CULTIVATION. 

To  cultivate  micro-organisms  artificially,  and,  in  the 
case  of  the  pathogenic  bacteria,  to  fulfil  the  second 
of  Koch's  postulates,  they  must  be  supplied  with 
nutrient  material  free  from  pre-existing  micro- 
organisms. Hitherto  various  kinds  of  nutrient 
liquids  have  been  employed,  and  in  many  cases  they 
still  continue  to  be  used  with  advantage,  but  as  a 
general  rule  they  have  been  in  a  great  measure 
supplanted  by  the  methods  of  cultivation  on  sterile 
solid  media  about  to  be  described.  The  advantages 
of  the  latter  method  are  obvious.  In  the  first  place, 
in  the  case  of  liquid  media,  in  spite  of  elaborate 
precautions  and  the  expenditure  of  much  labour  and 
time,  it  was  almost  impossible  or  extremely  difficult 
to  obtain  a  pure  culture.  If  a  drop  of  liquid  con- 
taining several  kinds  of  bacteria  be  introduced  into 
a  liquid  medium,  we  have  a  mixed  cultivation  from 
the  very  first.  If  in  the  struggle  for  existence  some 
bacteria  were  unable  to  develop  in  the  presence  ol 
others,  or  a  change  of  temperature  and  soil  allowed 


PREPARATION    OF   NUTRIENT    MEDIA.  63 

one  form  to  predominate  over  another,  then  one 
might  be  led  to  the  conclusion  that  many  bacteria 
were  but  developmental  forms  of  one  and  the  same 
micro-organism ;  while  possibly  the  contamination 
of  such  cultures  might  lead  to  the  belief  in  the  trans- 
formation of  a  harmless  into  a  pathogenic  bacterium. 
In  the  case  of  solid  cultivating  media,  on  the  other 
hand,  the  chance  of  contamination  by  gravitation  of 
germs  from  the  air  is  avoided  by  the  fact  that  test- 
tubes,  flasks,  etc.,  can  be  inverted  and  inoculated  from 
below.  The  secret  of  the  success  of  Koch's  method, 
however,  depends  upon  the  possibility,  in  the  case 
of  starting  with  a  mixture  of  micro-organisms,  of 
being  able  to  isolate  them  completely  one  from 
another,  and  to  obtain  an  absolutely  pure  growth 
of  each  cultivable  species.  When  sterile  nutrient 
gelatine  has  been  liquefied  in  a  tube  and  inoculated 
with  a  mixture  of  bacteria  in  such  a  way  that  the  in- 
dividual micro-organisms  are  distributed  throughout 
it,  and  the  liquid  is  poured  out  on  a  plate  of  glass  and 
allowed  to  solidify,  the  individual  bacteria,  instead 
of  moving  about  freely  as  in  a  liquid  medium,  are 
fixed  in  one  spot,  where  they  develop  individuals  of 
their  own  species.  In  this  way  colonies  are  formed 
each  possessing  its  own  characteristic  biological 
and  morphological  appearances.  If  an  adventi- 
tious germ  from  the  air  falls  upon  the  culture,  it 
also  grows  exactly  upon  the  spot  upon  which  it  fell, 
and  can  be  easily  recognised  as  a  stranger.  To 
maintain  the  individuals  isolated  from  one  another 


64  BACTERIOLOGY. 

during  their  growth,  and  free  from  contamination,  it 
is  only  necessary  to  thin  out  the  cultivation,  and  to 
protect  the  plates  from  the  air.  The  slower  growth 
of  the  micro-organisms  in  solid  media,  and  the 
greater  facility  afforded  thereby  for  examining  them 
at  various  intervals  and  stages  of  development,  is 
an  additional  point  in  favour  of  these  methods  ;  and 
the  characteristic  macroscopical  appearances  so 
frequently  assumed  are,  more  especially  in  the  case 
of  morphological  resemblance  or  identity,  of  the 
greatest  importance.  The  colonies  on  nutrient 
gelatine  (examined  with  a  low  power)  of  Bacillus 
anthracis  and  Proteus  mirabilis ;  the  naked  eye  ap- 
pearances in  test-tubes  of  nutrient  gelatine  of  the 
bacillus  of  mouse-septicaemia  (Figs.  114,  115),  and 
of  anthrax  (Fig.  107),  and  the  brilliant  and  curious 
growth  of  Bacterium  indicum  upon  nutrient  agar- 
agar  (Plate  II.,  Fig.  i),  may  be  quoted  as  examples 
in  which  the  appearances  in  solid  cultivations  are 
pathognomonic. 

SOLID  MEDIA 

(A)   PREPARATION    OF    STERILE    GELATINE-,    AND 

j  AGAR-AGAR-PEPTONE-BROTH. 

i 

Sterile  Gelatine-Peptone-broth,  or  Nutrient 
Gelatine,  is  prepared  as  follows : — Take  half  a 
kilogramme  of  beef  (one  pound),  as  free  as  possible 
from  fat.  Chop  it  up  finely,  transfer  it  to  a  flask 
or  cylindrical  vessel,  and  shake  it  up  well  with  a 


PREPARATION    OF   NUTRIENT   MEDIA.  65 

litre  of  distilled  water.  Place  the  vessel  in  an  ice- 
pail,  ice-cupboard,  or  in  winter  in  a  cold  cellar, 
and  leave  for  the  night.  Next  morning1  commence 
with  the  preparation  of  all  requisite  apparatus. 
Thoroughly  wash,  rinse  with  alcohol,  and  allow  to 
dry,  about  100  test-tubes.  Plug  the  mouth  of  the 
test-tubes  with  cotton  wool,  taking  care  that  the 
plugs  fit  firmly,  but  not  too  tightly.  Place  them 
in  their  wire  cages  in  the  hot-air  steriliser  to  be 
heated  for  an  hour  at  a  temperature  of  150°  C. 
In  the  same  manner  cleanse  and  sterilise  several 
flasks  and  a  small  glass  funnel.  In  the  meantime 
the  meat  infusion  must  be  again  well  shaken, 
and  the  liquid  portion  separated  by  filtering  and 
squeezing  through  a  linen  cloth.  The  red  juice 
thus  obtained  must  be  brought  up  to  a  litre  by 
again  transferring  it  to  a  large  measuring  glass 
and  adding  distilled  water.  It  is  then  poured  into 
a  sufficiently  large  and  strong  beaker;  and  set  aside 
after  the  addition  of 

10  grammes  of  peptonum  siccum, 
5  grammes  of  common  salt. 
100  grammes  of  best  gelatine. 

In  about  half-an-hour  the  gelatine  is  sufficiently 
softened,  and  subsequent  gently  heating  in  a  water- 
bath  causes  it  to  be  completely  dissolved.  The 
danger  of  breaking  the  beaker  may  be  avoided  by 
placing  a  cloth  several  times  folded  at  the  bottom 
of  the  water-bath. 


66  BACTERIOLOGY. 

The  next  process  requires  the  greatest  care  and 
attention.  Some  micro-organisms  grow  best  in  a 
slightly  acid,  others  in  a  neutral  or  slightly  alkaline 
medium.  For  example,  for  the  growth  and  cha- 
racteristic appearances  of  the  comma  bacillus  of 
Asiatic  cholera  a  faintly  alkaline  soil  is  absolutely 
essential.  This  slightly  alkaline  medium  will  be 
found  to  answer  best  for  most  micro-organisms,  and 
may  be  obtained  as  follows  : — 

With  a  clean  glass  rod  dipped  into  the  mixture, 
the  re-action  upon  litmus  paper  may  be  ascer- 
tained, and  a  concentrated  solution  of  carbonate  of 
soda  must  be  added  drop  by  drop,  until  red  litmus 
paper  becomes  faintly  blue.  If  it  has  been  made 
too  alkaline  it  can  be  neutralised  by  the  addition 
of  lactic  acid. 

Finally,  the  mixture  is  heated  for  an  hour 
in  the  water-bath  and  filtered  while  hot.  For  the 
filtration  the  hot-water  apparatus  can  be  used 
with  advantage,  furnished  with  a  filter  of  Swedish 
paper  made  in  the  following  way. 

About  eighteen  inches  square  of  the  best  and 
stoutest  filtering  paper  is  first  folded  in  the  middle, 
and  then,  as  in  Fig.  26,  creased  into  sixteen  folds. 
The  filter  is  made  to  fit  the  glass  funnel  by 
gathering  up  the  folds  like  a  fan,  and  cutting  off 
the  superfluous  part.  The  creasing  of  each  fold 
should  be  made  firmly  to  within  half  an  inch  of 
the  apex  of  the  filter,  which  part  is  to  be  gently 
inserted  into  the  tube  of  the  funnel.  To  avoid 


PREPARATION    OF   NUTRIENT    MEDIA.  67 

bursting  the  filter  at  the  point,  the  broth  when 
poured  out  from  the  flask  should  be  directed 
against  the  side  of  the  filter  with  a  glass  rod. 
During  filtration  the  funnel  should  be  covered  over 
with  a  circular  plate  of  glass,  and  the  process  of 
filtration  must  be  repeated,  if  necessary,  until  a 
pale,  straw-coloured,  perfectly  transparent  filtrate 
results. 

The    sterilised    test-tubes    are    filled    for   about 
a  third  of  their  depth  by  pouring  in  the  gelatine 


Fig.  26.— METHOD  OF  MAKING  A  FOLDED  FILTER. 

carefully  and  steadily,  or  by  employing  a  small 
sterilised  glass  funnel.  The  object  of  this  care 
is  to  prevent  the  mixture  touching  the  part  of 
the  tube  with  which  the  plug  comes  into  contact ; 
otherwise,  when  the  gelatine  sets,  the  cotton  wool 
adheres  to  the  tube,  and  becomes  a  source  of 
embarrassment  in  subsequent  procedures.  As  the 
tubes  are  filled  they  are  placed  in  the  test-tube 
basket,  and  must  then  be  sterilised.  They  are 
either  lowered  into  the  steam  steriliser,  when  the 


68  BACTERIOLOGY. 

thermometer  indicates  ico°  C.,  for  twelve  minutes 
for  four  or  five  successive  days ;  or  they  may  be 
transferred  to  the  test-tube  water-bath  and  heated 
for  an  hour  a  day  for  three  successive  days. 

If  the  gelatine  shows  any  turbidity  after  these 
processes,  it  must  be  poured  back  from  the  test- 
tubes  into  a  flask  and  boiled  up  for  ten  minutes, 
after  the  addition  of  the  white  and  shell  of  an  egg- 
beaten  up  together.  It  is  once  more  filtered,  and 
the  processes  of  sterilisation  just  described  must 
be  repeated. 

Sterile  Agar  -  Agar  -  Peptone  -  broth,  or 
Nutrient  Agar-Agar. — Agar-Agar  has  the  ad- 
vantage of  remaining  solid  up  to  a  temperature  of 
about  45°.  The  preparation  of  a  sterile  nutrient 
jelly  is  conducted  on  much  the  same  principles  as 
those  already  described.  Instead,  however,  of  100 
grammes  of  gelatine,  only  about  20  grammes 
of  agar-agar  are  employed  (1*5 — 2  per  cent.),  and 
to  facilitate  its  solution  it  must  be  allowed  to 
soak  in  salt-water  overnight.  For  the  filtration, 
flannel  is  substituted  for  filter  paper,  or  may  be 
used  in  combination  with  the  latter.  The  hot- 
water  apparatus  is  invariably  employed,  unless, 
to  accelerate  the  process,  the  glass  funnel  and 
receiver  are  bodily  transferred  to  the  steam 
steriliser.  If  the  conical  cap  cannot  be  replaced, 
cloths  laid  over  the  mouth  of  the  steriliser  must  be 
employed  instead.  It  may  be  necessary  to  repeat 
the  process  of  filtration,  but  it  must  not  be 


TEST-TUBE    CULTIVATIONS.  69 

expected  that  such  a  brilliant  transparency  can  be 
obtained  as  with  gelatine.  The  final  result,  when 
solid,  should  be  colourless  and  clear,  but  if  only 
slightly  milky  it  may  still  be  employed. 

After  the  final  treatment  in  the  steam  steriliser 
some  of  the  tubes  may  be  placed  in  the  blood- 
serum  apparatus,  and  left  to  gelatinise  with  an 
oblique  surface.  A  little  liquid  gradually  collects 
at  the  bottom  of  the  surface,  being  expressed  by  the 
contraction  of  the  nutrient  jelly. 

(B)    METHODS    OF    EMPLOYING    NUTRIENT    JELLY    IN 
TEST-TUBE-    AND    PLATE-CULTIVATIONS. 

Test-Tube-Cultivations. — To  inoculate  test- 
tubes  containing  nutrient  jelly,  the  cotton  wool  plug 
must  be  twisted  out,  by  which  means  any  adhesions 
that  may  exist  are  broken  down.  A  sterilised 
needle  charged  with  the  blood,  pus,  etc.,  containing 
the  micro-organisms,  or  with  a  colony  from  a 
plate-culture,  is  steadily  thrust  once,  and  once 
only,  into  the  nutrient  jelly.  The  tube  should  be 
held  with  its  mouth  downwards,  to  avoid,  as  far 
as  possible,  accidental  contamination  from  the 
gravitation  of  germs  in  the  air,  and  the  plug, 
which  has  been  removed  with  the  thumb  and  index 
finger  of  the  right  hand  and  held  between  the 
fourth  and  fifth  fingers  of  the  left,  is  replaced 
as  rapidly  as  possible  (Fig.  27). 

The  chances  of  error  arising  from  contamination 


BACTERIOLOGY. 


of  the  cultivations  are  further  reduced  by  avoiding 
draughts  at  the  time  of  inoculation,  and  it  is  best 
that  these  manipulations  should  be  carried  on  in  a 
quiet  room  in  which  the  tables  and  floor  are  wiped 
with  damp  cloths,  rather  than  in  a  laboratory  in 
which  the  air  becomes  charged  with  germs  through 

constant  sweeping  and  dust- 
ing, and  the  entrance  and 
exit  of  classes  of  students. 
In  conducting  any  investi- 
gation a  dozen  or  more 
tubes  should  be  inoculated, 
and  if  by  chance  an  ad- 
ventitious germ,  in  spite  of 
these  precautions,  gain  an 
entrance,  the  contaminated 
tube  can  be  rejected  and 
the  experiments  continued 
with  the  remaining  pure 
cultivations. 

Where,  however,  one  tube  is  inoculated  from 
another  containing  a  liquid  medium,  as  in  the  process 
of  preparing  plate-cultures,  or  where  a  culture  is 
made  from  a  tube  in  which  the  growth  has  liquefied 
the  gelatine,  it  is  obvious  that  the  tubes  cannot  be 
inverted,  and  they  must  then  be  held  and  inoculated 
as  in  Fig.  28.  To  inoculate  those  tubes  of  nutrient 
agar-agar  which  have  been  gelatinised  obliquely,  the 
sterilised  needle  with  the  material  to  be  cultivated 
is  streaked  over  the  surface  from  below  upwards. 


FIG,  27. 

METHOD  OF  INOCULATING 
A  TEST-TUBE  CONTAINING 
STERILE  NUTRIENT  JELLY. 


TEST-TUBE    CULTIVATIONS.  J  I 

Examination    of   test-tube    cultivations. — 

The  appearances  produced  by  the  growths  in  test- 
tubes  can  be  in  most  cases  sufficiently  examined 
with  the  naked  eye.  The  illustrations  in  the  ac- 
companying plates  (Plates  II.,  III.,  IV.,  V.,  VIII., 
XIII.,  XIV.,  XXIV.)  are  given  as  examples  of  the 
various  changes  produced  in  the  nutrient  media. 
In  some  cases  the  jelly  is  partially  or  completely 
liquefied,  while  in  others  it  remains  solid.  The 
growths  may  be  abundant  or  scanty,  coloured  or 
colourless.  The  nutrient  jelly  may  itself  be  tinged 
or  stained  with  products  resulting  from  the  growth 
of  the  organisms.  When  liquefaction  slowly  takes 
place  in  the  needle  track,  or  the  organism  grows 
without  producing  this  change,  the  appearances 
which  result  are  often  very  delicate,  and  in  some 
cases  very  characteristic.  The  appearance  of  a 
simple  white  thread,  of  a  central  thread  with 
branching  transverse  filaments,  of  a  cloudiness,  or 
of  a  string  of  beads  in  the  track  of  the  needle,  may 
be  given  as  examples.  In  such  cases  much  may  be 
learnt  by  examining  the  growth  with  a  magnifying 
glass.  Here,  however,  a  difficulty  is  encountered, 
for  the  cylindrical  form  of  the  tube  so  distorts  the 
appearance  of  its  contents,  that  the  examination  is 
rendered  somewhat  difficult.  To  obviate  this,  the 
following  very  simple  contrivance  may  be  employed 
with  advantage. 

Cheshire's  Trough. — This  consists  of  a  rect- 
angular vessel,  four  inches  in  height,  two  inches  in 


7  2  BACTERIOLOGY. 

width,  and  one  inch  in  depth.  It  may  be  easily 
constructed  by  cementing  together  two  slips  of  glass 
to  form  the  back  and  front,  with  three  slips  of  stout 
glass  with  ground  edges  forming  the  sides  and 
base.  The  front  may  be  constructed  of  thin  glass, 
and  the  base  of  the  vessel  made  to  slope  so  that  the 
test-tube  when  placed  in  the  trough  has  a  tendency 
to  be  near  the  front.  The  trough  is  filled  with  a 
mixture  of  the  same  refractive  index  as  the  nutrient 
gelatine.  The  latter  has  a  refractive  index  rather 
higher  than  water,  which  is  about  1*333;  alcohol 
has  a  refractive  index  of  1*374.  The  trough  is 
rilled  with  water,  and  alcohol  is  then  added  until  the 
proper  density  is  reached.  The  test-tube  is  placed 
in  the  trough,  and  held  in  position  by  means  of  a 
clip.  The  trough  can  be  fixed  on  the  inclined  stage 
of  the  microscope,  and  the  contents  of  the  tube 
conveniently  examined  with  low  power  objectives. 

PLATE-CULTIVATIONS. 

The  key  to  the  success  of  Koch's  method  of 
cultivation  on  solid  media  consists  in  the  employ- 
ment of  plate-cultivations.  By  this  means,  as 
has  already  been  mentioned  (p.  63),  a  mixture  of 
bacteria,  whether  it  be  in  fluids,  excreta,  or  in 
artificial  cultivations,  can  be  so  treated  that  the 
different  species  are  isolated  one  from  the  other, 
and  perfectly  pure  cultivations  of  each  of  the 
cultivable  bacteria  in  the  original  mixture  esta- 
blished in  various  nutrient  media.  We  are  enabled 


PLATE-CULTIVATIONS.  73 

also  to  examine  under  a  low  power  of  the 
microscope  the  individual  colonies  of  bacteria, 
and  to  distinguish,  by  their  characteristic  appear- 
ances, micro-organisms  which,  in  their  individual 
form,  closely  resemble  one  another,  or  are  even 
^identical.  The  same  process,  with  slight  modifica- 
tion, is  also  employed  in  the  examination  of  air, 
soil,  and  water,  as  will  be  referred  to  later. 

The  preparation  of  plate-cultivations,  therefore, 
must  be  described  in  every  detail,  and  to  take 
an  example,  we  will  suppose  that  a  series  of  plates 
are  to  be  prepared  from  a  test-tube-cultivation. 

Arrangement  of  Levelling  Apparatus. — In 
order  to  spread  out  the  liquid  jelly  evenly  on  the 
surface  of  a  glass  plate,  and  hasten  its  solidifica- 
tion, it  is  necessary  to  place  the  glass  plate  upon  a 
level  and  cool  surface.  This  is  obtained  in  the 
following  manner : — Place  a  large  shallow  glass 
dish  upon  a  tripod  stand,  and  fill  it  to  the  brim 
with  cold  water;  carefully  cover  the  dish  with  a 
slab  of  plate  glass,  or  a  pane  of  window  glass,  and 
level  it  by  placing  the  spirit-level  in  the  centre  and 
adjusting  the  screws  of  the  tripod.  Replace  the 
level  by  a  piece  of  filter  paper,  the  size  of  the  glass 
plates  to  be  employed,  and  cover  it  with  a  shallow 
bell  glass  (Fig.  8). 

Sterilisation  of  Glass  Plates. — The  glass 
plates  are  sterilised  by  filling  the  iron  box  (p.  27), 
and  placing  it  in  the  hot-air  steriliser,  at  150°  C., 
from  one  to  two  hours.  As  these  plates  are  used 


74  BACTERIOLOGY. 

also  for  other  purposes,  a  quantity  ready  sterilised 
should  always  be  kept  in  the  box. 

Preparation  of  Damp  Chambers. — The  damp 
chambers  for  the  reception  of  the  inoculated  plates 
are  prepared  thus  : — Thoroughly  cleanse  and  wash 
out  with  (i-iooo)  sublimate  solution  a  shallow 
glass  dish  and  bell  (Fig.  7).  Cut  a  piece  of  filter 
paper  to  line  the  bottom  of  the  glass,  dish,  and 
moisten  it  with  the  same  solution. 

In  a  glass  beaker  or  an  ordinary  glass  tumbler, 
with  a  pad  of  cotton  wool  at  the  bottom,  place 
the  tube  containing  the  cultivation,  the  three  tubes 
to  be  inoculated,  and  three  glass  rods  which  have 
been  sterilised  by  heating  in  the  flame  of  a  Bunsen 
burner.  Provide  yourself  with  a  strip  of  paper  01 
a  large  label,  a  pencil,  a  pair  of  forceps,  and 
inoculating  needles.  All  is  now  ready  at  hand  to 
commence  the  inoculation  of  the  tubes. 

Method  of  Inoculating  the  Test-tubes. 
Liquefy  the  gelatine  in  the  three  tubes  by  placing 
them  in  a  beaker  containing  water  at  30°  C.,  or 
by  gently  warming  them  in  the  flame  of  the  Bunsen 
burner.  Keep  the  tubes,  both  before  and  after  the 
inoculation,  in  the  warm  water,  to  maintain  the 
gelatine  in  a  state  of  liquefaction.  Hold  the  tube 
containing  the  cultivation  and  a  tube  of  the 
liquefied  gelatine  (to  be  called  the  "  original ") 
as  nearly  horizontal  as  possible  between  the  thumb 
and  index  finger  of  the  left  hand.  With  the  finger 
and  thumb  of  the  right  hand  loosen  the  plugs  ot 


PLATE-CULTIVATIONS.  7  c 

the  tubes.  Take  the  sterilised  ose  in  the  right 
hand  and  hold  it  like  a  pen.  Remove  the  plug 
from  the  culture-tube  by  using  the  fourth  and  fifth 
fingers  of  the  right  hand  as  forceps,  and  place  it 
between  the  fourth  and  fifth  fingers  of  the  left. 
Remove  the  plug  of  the  "  original  "  in  the  same  way, 
placing  it  between  the  third  and  fourth  fingers  of  the 
left  hand  (Fig.  28).  With  the  ose  take  up  a  droplet 


FIG.  28.— METHOD  OF  INOCULATING  TEST-TUBES  IN  THE  PREPARATION 
OF  PLATE-CULTIVATIONS. 

of  the  cultivation  and  inoculate  the  *'  original," 
twisting  the  ose  several  times  in  the  liquid  gelatine. 
Replace  the  plugs  and  set  aside  the  cultivation. 
Hold  the  freshly  inoculated  tube  between  the  fore- 
finger and  thumb  of  either  hand,  almost  horizontally, 
then  raise  it  to  the  vertical,  so  that  the  liquid  gelatine 
gently  flows  back.  By  repeating  this  motion  and 
rolling  the  tube  between  the  fingers  and  thumbs  the 
micro-organisms  which  have  been  introduced  are 


76  BACTERIOLOGY. 

distributed  throughout  the  gelatine.  Any  violent 
shaking,  and  consequent  formation  of  bubbles, 
must  be  carefully  avoided.  From  the  so-called 
"original"  inoculate  in  the  same  manner  afresh 
tube  of  liquefied  gelatine,  introducing  into  it 
three  droplets  with  a  sterilised  ose.  This  tube  is 
then  called  the  "  first  attenuation,"  or  No.  i.  After 
treating  No.  i  as  has  been  already  described  in  the 
case  of  the  original,  the  same  process  is  repeated 
with  a  third  tube,  which  is  inoculated  in  the  same 
way  from  No.  i.  This  is  the  "second  attenua- 
tion" or  No.  2,  and  in  some  cases  a  "third  atten- 
uation "  is  carried  out  from  No.  2.  The  last  tube 
must  be  inoculated  in  different  ways,  according  to 
experience  for  different  micro-organisms.  Some- 
times a  sufficient  separation  of  the  micro-organisms 
is  attained  by  inoculating  the  last  tube  with  a  fine 
straight  needle  dipped  from  one  into  the  other 
from  three  to  five  times. 

The  next  process  consists  in  pouring  out  the 
gelatine  on  a  glass  plate  and  allowing  it  to  solidify. 

Preparation  of  the  Gelatine-plates. — The 
directions  to  be  observed  in  pouring  out  the  gelatine 
are  as  follows  : — 

Place  the  box  containing  sterilised  plates  horizon- 
tally, and  so  that  the  cover  projects  beyond  the  edge 
of  the  table  ;  remove  the  cover,  and  withdraw  a 
plate  with  sterilised  forceps  ;  hold  it  between  the 
finger  and  thumb  by  opposite  margins,  rapidly 
transfer  it  to  the  filter  paper  under  the  bell-glass,  and 


PLATE-CULTIVATIONS.  7  7 

quickly  replace  the  cover  of  the  box.  On  removing 
the  plug  from  "the  original,"  an  assistant  raises 
the  bell-glass,  and  the  contents  of  the  tube  are 
poured  on  to  the  plate ;  with  a  glass  rod  the  gelatine 
must  be  then  rapidly  spread  out  in  an  even  layer 
within  about  half  an  inch  of  the  margin  of  the 
plate.  The  assistant  replaces  the  bell-glass,  and  the 
gelatine  is  left  to  set.  Meanwhile  a  glass  bench  or 
metallic  shelf  is  placed  in  the  damp  chamber,  ready 
for  the  reception  of  the  plate-cultivation,  and  when 
the  gelatine  is  quite  solid  the  plate  is  quickly  trans- 
ferred from  under  the  bell-glass  to  the  damp  chamber; 
precisely  the  same  process  is  repeated  with  tubes  i 
and  2,  and  the  damp  chamber,  labelled  with  the 
details  of  the  experiment,  is  set  aside  for  the  colonies 
to  develop.  Not  only  plate- cultures  should  be  care- 
fully labelled  with  date  and  description,  but  the 
same  remark  applies  equally  to  all  preparations, 
tube- cultures,  potato-cultures,  drop-cultures,  etc. 
In  plate-cultivations  write  the  source  of  the  mate- 
rial, the  date,  and  the  number  of  inoculations;  for 
example,  thus  : — • 

Finkler' }  s  comma-bacilli. 

From  tube-cultivation  on    "  agar-agar,"   5th  Feb- 
ruary, 1885 

Lower  plate  (Orig.)      .     i  ose  from  cultivation. 
Middle  plate,  No.  i      .     3  osen  from  Orig. 
Upper  plate,  No.  2 .     .     3  osen  from  No.  i . 


78  BACTERIOLOGY. 

Corresponding  with  the  fractional  cultivation  of 
the  micro-organisms  obtained  in  this  manner,  the 
colonies  will  be  found  to  develop  in  the  course  of  a 
day  or  two,  varying  with  the  temperature  of  the 
room.  The  lower  plate  will  contain  a  countless 
number  of  colonies  which,  if  the  micro-organism 
liquefies  gelatine,  speedily  commingle,  and  produce, 
in  a  very  short  time,  a  complete  liquefaction  of  the 
whole  of  the  gelatine.  On  the  middle  plate,  with 
the  first  attenuation,  the  colonies  will  also  be  very 
numerous,  but  retain  their  isolated  position  for  a 
longer  time ;  while  on  the  uppermost  plate,  the 
second  attenuation,  the  colonies  are  completely 
isolated  from  one  another,  with  an  appreciable 
surface  of  gelatine  intervening  [Plates  VI.  and  VII.]. 

Examination  of  Plate  -  cultivations. — The 
macroscopical  appearances  of  the  colonies  are  best 
studied  by  placing  the  plate  on  the  slab  of  blackened 
glass,  or  on  the  porcelain  slab  if  the  colonies  are 
coloured. 

To  examine  the  microscopical  appearances  a 
selected  plate  is  placed  upon  the  stage  of  the 
microscope ;  it  is  better  to  have  a  larger  stage  than 
usual  for  this  purpose.  The  smallest  diaphragm  is 
employed,  and  the  appearances  studied  principally 
with  a  low  power.  These  appearances  should  be 
carefully  noted,  and  a  rapid  sketch  of  the  co^ny 
made.  The  morphological  characteristics  of  the 
micro-organisms  of  which  the  colony  is  formed  can 
then  be  examined  in  the  following  way.  A  small 


PLATE-CULTIVATIONS. 


79 


6'se,  or  a  platinum  needle  bent  at  the  extremity 
into  a  miniature  hook,  is  held  like  a  pen,  and  the 
hand  steadied  by  resting  the  little  finger  on  the 
stage  of  the  microscope.  The  extremity  of  the  needle 
is  steadily  directed  to  the  space  between  the  lens 
and  the  gelatine  without  touching  the  latter,  until, 


FIG.  29. — MICROSCOPICAL  EXAMINATION  OF  COLONIES  ON  PLATE- 
CULTIVATIONS,  AND  METHOD  OF  ISOLATING  COLONIES  BY 
INOCULATION  OF  TEST-TUBES. 

on  looking  through  the  microscope,  it  can  be  seen 
in  the  field,  above  or  by  the  side  of  the  colony  under 
examination  (Fig.  29).  The  needle  is  then  dipped 
into  the  colony,  steadily  raised,  and  withdrawn. 
Without  removing  the  eye  from  the  microscope 
this  manipulation  can  be  seen  to  be  successful  by 
the  colony  being  disorganised  or  completely  re- 


8O  BACTERIOLOGY. 

moved  from  the  gelatine.  It  is,  however,  not  easy 
to  be  successful  at  first,  but  with  practice  this  can 
be  accomplished  with  rapidity  and  precision.  A 
cover-glass-preparation  is  then  made  in  the  manner 
already  described,  viz.,  by  rubbing  the  extremity 
of  the  needle  on  a  perfectly  clean  cover-glass  and 
examining  by  Babes'  rapid  method,  or  by  thinning 
out  the  micro-organisms  in  a  droplet  of  sterilised 
water  previously  placed  on  the  cover  glass,  drying, 
passing  three  times  through  the  flame,  and  staining 
with  a  drop  of  fuchsine. 

Inoculations  should  be  made  in  test-tubes  of 
nutrient  gelatine  and  agar-agar,  from  the  micro- 
organisms transferred  to  the  cover-glass  before 
it  is  dried  and  stained,  from  any  remnants  of  the 
colony  which  was  examined,  or  from  other  colonies 
bearing  exactly  similar  appearances.  In  this  way 
pure  cultivations  are  established,  and  the  macro- 
scopical  appearances  of  the  growth  in  test-tubes 
can  be  studied.  The  plates  should  be  replaced 
in  the  damp  chambers  as  soon  as  possible ;  drying 
of  the  gelatine,  or  contamination  with  micro- 
organisms gravitating  from  the  air  during  their 
exposure,  may  spoil  them  for  subsequent  examina- 
tion. Nutrient  agar-agar  can  also  be  employed 
for  the  preparation  of  plate- cultivations,  but  it  is 
much  more  difficult  to  obtain  satisfactory  results. 
The  test-tubes  of  nutrient  agar-agar  must  be 
placed  in  a  beaker  with  water  and  heated  until  the 
agar-agar  is  completely  liquefied.  The  gas  is  then 


PLATE-CULTIVATIOXS.  8 1 

turned  down  and  the  temperature  of  the  water 
allowed  to  fall  until  the  thermometer  stands  just 
above  50°  C.  The  water  must  be  maintained  at 
this  temperature,  and  the  test-tubes  must  be  in  turn 
rapidly  inoculated  and  poured  out  upon  the  glass 
plates,  as  already  described.  Glass  plates  may 
also  be  employed  in  a  much  simpler  method.  The 
nutrient  jelly  is  liquefied,  poured  out,  and  allowed 
to  set.  A  needle  charged  with  the  material  to  be 
inoculated  is  then  streaked  in  lines  over  the  surface 
of  the  jelly.  This  method  is  of  especial  value  in 
inoculating  different  organisms  side  by  side,  and 
watching  the  effect  of  one  upon  the  other,  or  a 
micro-organism  in  this  way  may  be  sown  upon 
gelatine  which  has  been  already  altered  by  the 
growth  of  another  micro-organism ;  the  change 
produced  in  the  gelatine,  as  in  the  case  of  the 
fluorescing  bacillus,  extending  far  beyond  the 
limits  of  the  growth  itself  (Plate  VIII.,  Fig.  3). 
Nutrient  jelly  may  also  be  spread  out  on  steri- 
lised microscopic  slides  and  inoculated  as  just 
described,  or  cultivations  may  be  made  in  shallow 
glass  dishes,  glass  capsules,  etc.,  which  must  be 
sterilised  on  the  principles  already  laid  down,  and 
after  inoculation  placed  in  damp-chambers  for  the 
growths  to  develop. 


82  BACTERIOLOGY. 


(C)  PREPARATION  AND  EMPLOYMENT  OF  STERILISED 
POTATOES,  POTATO -PASTE,  BREAD-PASTE,  VEGE- 
TABLES, FRUIT,  AND  WHITE  OF  EGG. 

Potato  Cultivations. — Sterilised  potatoes  form 
an  excellent  medium  for  the  cultivation  of  many 
micro-organisms,  more  especially  the  chromogenous 
species.  Potato-cultivations  also  give  in  some  cases 
very  characteristic  appearances,  which  are  of  value 
in  distinguishing  bacteria  which  possess  morphologi- 
cal resemblances  [Plates  IX.  and  X.] 

Preparation  of  Sterilised  Potatoes. — Potatoes, 
preferably  smooth- skinned,  which  are  free  from 
"  eyes  "  and  rotten  spots,  should  be  selected.  If  they 
cannot  be  obtained  without  eyes  and  spots,  these 
must  be  carefully  picked  out  with  the  point  of  a 
knife  with  as  little  destruction  of  the  surface  as 
possible.  The  potatoes  are  well  scrubbed  with  a 
stiff  brush  and  allowed  to  soak  in  sublimate  solution 
for  half  an  hour.  They  are  then  transferred  to  the 
potato-receiver  and  steamed  in  the  steam-steriliser 
for  twenty  minutes  to  half  an  hour,  varying  accord- 
ing to  the  size  of  the  potatoes.  When  cooked,  the 
potato-receiver  is  withdrawn  and  left  to  cool,  the 
potatoes  being  retained  in  it  until  required  for  use. 

Damp  chambers  are  prepared  ready  for  their 
reception,  the  vessels  being  cleansed  and  washed 
with  sublimate  as  described  for  plate-cultivations. 
Small  glass  dishes  of  the  same  pattern  as  the 


PREPARATION    OF   NUTRIENT   MEDIA.  83 

large  ones  (Fig.  13)  may  be  employed  for  single 
halves  of  potatoes.  Potato  knives  and  several 
scalpels  which  have  been  sterilised  in  an  Israel's 
case  by  heating  them  in  the  hot-air  steriliser  to  150° 
for  one  hour,  must  be  ready  to  hand.  The  potato 
knives  may  also  be  sterilised  by  heating  them  in 
the  flame  of  a  Bunsen  burner  and  placing  them 
on  their  backs  with  their  blades  projecting  over 
the  edge  of  the  table.  Scalpels  may  be  sterilised 
in  the  same  way  and  laid  upon  a  sterilised  glass 
plate  and  covered  with  a  bell-glass.  It  must  not 
be  forgotten,  however,  that  heating  the  blades  in 
the  flame  destroys  the  temper  of  the  steel,  and 
therefore  knives  and  other  instruments  should 
preferably  be  sterilised  in  the  hot-air  steriliser,  en- 
closed in  an  Israel's  case,  or  simply  enveloped 
in  cotton  wool. 

Inoculation  of  Potatoes. — The  coat  sleeves 
should  be  turned  back,  and  the  hands,  after  a 
thorough  washing  with  good  lathering  soap,  be 
dipped  in  sublimate  solution.  An  assistant  opens 
the  potato  receiver,  and  a  potato  is  selected, 
and  held  between  the  thumb  and  index  finger 
of  the  left  hand  (Fig.  30).  With  the  knife  held  in 
the  right  hand,  the  potato  is  almost  completely 
divided  in  the  direction  which  will  give  the  largest 
surface.  The  assistant  raises  the  cover  of  the 
damp  chamber,  and  the  potato  is  introduced, 
and  while  withdrawing  the  knife,  allowed  to  fall 
apart.  The  cover  is  quickly  replaced,  and  another 


84  BACTERIOLOGY. 

potato  treated  in  the  same  way  is  placed  in  the 
same  damp  chamber.  The  four  halves  are  then 
quite  ready  for  inoculation.  As  an  extra  pre- 
caution the  left  hand  is  again  dipped  in  sublimate, 
and  one  half  of  a  potato  is  taken  up  between  the 
tips  of  the  thumb  and  index  finger,  care  being 
taken  to  avoid  touching  the  cut  surface.  Holding 


FIG.  30.— METHOD  OF  DIVIDING  POTATOES. 

it  with  its  cut  surface  vertical,  a  small  portion 
of  the  substance  to  be  inoculated  is  placed  on 
the  centre  with  a  sterilised  needle  or  ose.  With 
a  sterilised  scalpel  the  inoculated  substance  is 
rapidly  spread  over  the  surface  of  the  potato 
with  the  flat  of  the  blade  to  within  a  quarter  of 
an  inch  of  the  margin,  and  the  potato  is  then  as 


PREPARATION    OF   NUTRIENT   MEDIA.  85 

quickly  as  possible  replaced  in  the  moist  chamber. 
With  another  sterilised  scalpel  a  small  portion  of 
the  potato  from  the  inoculated  surface  of  the  first 
half  is  in  the  same  way  spread  over  the  surface  of 
the  second  half,  this  forming  as  in  plate-cultiva- 
tions a  "  first  attenuation."  Exactly  the  same  is 
repeated  with  a  third  potato,  and  even  a  fourth,  so 
that  a  still  further  attenuation  or  fractional  culti- 
vation of  the  micro-organisms  may  be  obtained.  In 
some  cases  it  is  necessary  to  place  the  cultures  in 
the  incubator,  others  grow  very  well  at  the  tempera- 
ture of  the  room.  As  in  plate-cultivations,  the  potato 
may  also  be  inoculated  by  simply  streaking  it  in 
lines  with  a  needle  charged  with  the  material  to  be 
inoculated. 

POTATO-PASTE. 

Potato-paste  is  sometimes  employed  where  it 
is  desirable  to  obtain  an  extensive  growth  of  certain 
bacteria.  The  potatoes  are  boiled  for  an  hour, 
and  the  floury  centre  squeezed  out  of  their  skins. 
This  is  then  mashed  up  with  sufficient  sterilised 
water  to  produce  a  thick  paste,  and  is  heated  in 
the  steam  steriliser  for  half  an  hour  for  three 
successive  days. 

BREAD-PASTE. 

Some  micro-organisms,  more  especially  mould 
fungi,  grow  very  well  on  bread- paste.  This  is 
prepared  by  removing  the  crust  from  a  stale  loaf 


£6  BACTERIOLOGY. 

of  bread  and  allowing  it  to  dry  in  the  oven.  It 
is  then  broken  up,  and  reduced  to  a  fine  powder 
with  a  pestle  and  mortar.  Small,  carefully  cleansed, 
conical,  or  globe-shaped  flasks  are  plugged  with 
cotton  wool  and  sterilised  in  the  oven.  When 
cool  a  small  quantity  of  the  powder  is  placed  in 
them,  and  sterilised  water  added  in  the  proportion 
of  one  part  for  every  four  of  the  powder.  The 
paste  is  sterilised  by  steaming  in  the  steriliser 
at  1 00°  C.  for  half  an  hour  for  three  successive 
days.  The  flasks  can  be  reversed,  and  are  inocu- 
lated in  the  usual  way  with  a  platinum  needle. 

CULTIVATIONS     ON     VEGETABLES,     FRUIT,      WHITE     OF 
EGG,      ETC. 

Boiled  carrots  and  other  vegetables,  and  various 
kinds  of  stewed  fruit,  are  also  occasionally  em- 
ployed for  the  cultivation  of  bacteria.  The  sterili- 
sation of  these  media  must  be  carried  out  on  the 
principles  already  explained.  White  of  egg  may 
be  poured  out  on  sterilised  glass  plates,  or  in 
shallow  glass  dishes,  boiled  in  the  steam-steriliser 
and  after  inoculation,  placed  in  a  damp  chamber. 

(D)     PREPARATION   AND    EMPLOYMENT    OF    STERILE 
BLOOD    SERUM. 

The  tubercle-bacillus,  the  bacillus  of  glanders,  and 
a  few  other  micro-organisms,  thrive  best  when  culti- 


PREPARATION    OF    NUTRIENT    MEDIA.  8? 

vated  on  solid  blood  serum.  This  medium  has  the 
additional  advantage  of  remaining  solid  at  all  tem- 
peratures. The  technique  required  for  its  preparation 
and  sterilisation  is  as  follows  : — Several  cylindrical 
vessels,  about  20  cm.  high,  are  thoroughly  washed 
with  sublimate  solution  (i-iooo),  and  then  with 
alcohol,  and  finally  rinsed  out  with  ether.  The 
ether  is  allowed  to  evaporate,  and  the  vessels  are 
then  ready  for  use.  The  skin  of  the  animal 
selected — calf,  sheep,  or  horse — is  washed  with 
sublimate  at  the  seat  of  operation,  and  the  bleeding 
is  performed  with  a  sterilised  knife.  The  first  jet 
of  blood  from  the  vein  is  rejected,  and  that  which 
follows  is  allowed  to  flow  into  the  vessels  until 
they  are  almost  full.  The  ground-glass  stoppers, 
greased  with  vaseline,  are  replaced,  and  the  vessels 
set  aside  in  ice,  as  quickly  as  possible,  for  from 
twenty-four  to  thirty  hours.  By  that  time  the 
separation  of  the  clot  is  completed,  and  the  clear 
serum  can  then  be  transferred  to  plugged  sterile 
test-tubes.  These  should  be  filled  with  a  sterilised 
pipette  for  about  a  third  of  their  length,  and  are 
then  placed  in  Koch's  slow  steriliser  with  the  tem- 
perature maintained  for  an  hour  at  58°  C.  The 
same  process  is  repeated  for  six  successive  days, 
the  temperature  on  the  last  day  being  gradually 
raised  to  60°.  This  completes  the  sterilisation,  but 
to  solidify  the  serum  it  is  necessary  to  arrange  the 
tubes  in  the  inspissator  at  the  angle  required.  The 
temperature  of  this  apparatus  is  kept  between  65° 


88  BACTERIOLOGY. 

and  68°  C.  Directly  solidification  takes  place  the 
tubes  must  be  removed,  and  they  should  then 
present  the  character  of  being  hard,  solid,  of  a  pale 
straw  colour,  and  transparent.  A  little  liquid 
collects  at  the  lowest  point,  and  the  serum  is  some- 
times milky  in  appearance  at  its  thickest  part.  The 
serum  may  not  only  be  employed  in  test-tubes,  but 
also  in  small  flasks,  glass  capsules,  or  other  vessels, 
all  of  which  must  be  cleansed  and  sterilised  in 
the  usual  way.  Hydrocele  fluid  and  other  serous 
effusions  may  be  prepared  in  the  same  manner,  or 
gelatine  may  be  added  to  the  serum  in  the  propor- 
tion of  5  per  cent. 

Inoculation  of  the  Tubes. — A  small  portion 
of  the  material  to  be  inoculated  is  taken  up  with 
a  sterilised  needle  or  ose,  and  drawn  in  lines  over 
the  sloping  surface  of  the  serum;  or  a  minute  piece 
of  tissue,  tubercle,  etc.,  may  be  introduced  into  the 
tube  and  deposited  on  the  surface  of  the  nutrient 
medium.  The  precautions  that  are  to  be  observed 
in  isolating  the  material  to  be  inoculated  will  be 
referred  to  later  (p.  112). 

LIQUID  MEDIA. 

(E)  PREPARATION  OF  STERILISED  BOUILLON,  LIQUID 
BLOOD  SERUM,  URINE,  MILK,  VEGETABLE  INFU- 
SIONS, AND  ARTIFICIAL  NOURISHING  LIQUIDS. 

Nutrient  liquids  are  still  largely  employed,  and 
by  some  observers  even  in  preference  to  the  solid 


LIQUID    MEDIA.  89 

media  advocated  by  Koch.  It  must  not  be  supposed, 
however,  that  the  methods  of  cultivation  in  liquids 
are  discarded  entirely  by  the  German  school,  for 
there  is  no  more  instructive  method  than  the 
employment  of  so-called  drop-cultures.  For  inocu- 
lation experiments  where  the  presence  of  gelatine 
is  undesirable,  for  studying  the  physiology  and 
chemistry  of  bacteria  and  where  for  any  object 
a  rapid  growth  of  micro-organisms  is  necessary, 
the  employment  of  liquid  media  is  not  only  advis- 
able, but  is  absolutely  necessary.  Liquid  media 
comprise  two  distinct  groups — natural  and  artificial. 
The  natural  group  includes  meat  broths,  blood, 
urine,  milk,  and  vegetable  infusions ;  the  artificial 
are  solutions  built  up  from  a  chemical  formula 
representing  the  essential  food  constituents. 


NATURAL  MEDIA. 

Bouillon. — A  broth  or  bouillon  of  beef,  pork, 
or  chicken  may  be  made  in  the  same  manner  as 
described  for  the  preparation  of  gelatine-peptone- 
broth,  with  simply  omission  of  the  gelatine.  After 
the  neutralisation  with  carbonate  of  soda  solution 
drop  by  drop,  the  flask  of  broth  is  placed  in  the 
steam  steriliser  for  half  an  hour  at  100°  C.  A  clear 
liquid  results  on  filtration,  which  is  transferred  to 
plugged  sterilised  flasks  or  test-tubes,  and  sterilisa- 
tion effected  by  exposing  them  in  the  steam  steriliser 


gO  BACTERIOLOGY. 

for  half  an  hour  at  100°  C.  for  two  or  three  succes- 
sive days. 

Liquid  Blood  Serum. — The  preparation  of 
sterile  blood  serum  has  already  been  described.  It 
may  be  used  for  cultivation,  especially  in  the  form 
of  drop- cultivations,  before  the  final  treatment  by 
which  it  is  solidified.  Hydrocele  fluid,  peritonitic 
and  pleuritic  effusions,  can  also  be  employed  after 
sterilisation  in  the  steam  steriliser.  The  fluid  should 
be  withdrawn  with  a  sterilised  trocar  and  canula, 
and  received  into  plugged  sterilised  flasks. 

Urine. — In  order  to  obtain  urine  free  from  micro- 
organisms the  following  precautions  must  be  ob- 
served : — The  orifice  of  the  urethra  must  be 
thoroughly  cleansed  with  sublimate  solution.  The 
first  jet  of  urine  should  be  rejected,  and  the  rest 
received  into  sterilised  vessels,  which  must  be  quickly 
closed  with  sterile  plugs.  If  these  precautions  be  not 
attended  to  the  urine  must  be  rendered  sterile  by 
the  means  described  for  the  sterilisation  of  bouillon. 

Milk. — If  milk  has  been  drawn  into  sterile  flasks 
after  thoroughly  cleansing  and  disinfecting  the 
teats  and  hands,  it  may  be  kept  without  change. 
If  procured  without  these  precautions  it  must  be 
steamed  in  the  steriliser  for  half  an  hour  for  five 
successive  days. 

Vegetable  Infusions.  —  Infusions  of  hay, 
cucumber,  and  turnip  are  used  for  special  pur- 
poses, and  more  rarely  decoctions  of  plums,  raisins, 
malt,  and  horse-dung.  They  are  mostly  prepared 


LIQUID    MEDIA.  gi 

by  boiling  with  distilled  water,  after  maceration  for 
several  hours.  The  filtrate  is  received  into  sterile 
flasks  and  sterilised  in  the  usual  way  in  the  steam 
steriliser. 

ARTIFICIAL  FLUIDS. 

Pasteur's  Fluid. — This  solution  is  prepared  by 
mixing  the  ingredients  in  the  following  propor- 
tions : — 

Distilled  water  ,  .  .  .100 
Pure  cane  sugar  .  .  .  .  10 
Ammonium  tartrate  i 

Ash  of  yeast -075 

Cohn-Mayer  Fluid. — Mayer's  modification  of 
the  nourishing  fluid  employed  by  Cohn  is  as 
follows  : — 

Distilled  water         ....  20 

Phosphate  of  potassium  .         .         .  'i 

Sulphate  of  magnesium  .         .         .  'i 

Tribasic  calcium  phosphate     .         .  *oi 

Ammonium  tartrate         .         .         .  '2 

(F)    METHODS    OF    STORING    AND    EMPLOYING    LIQUID 

MEDIA;  LISTER'S  FLASKS,  AITKEN'S  TEST-TUBES, 
STERNBERG'S  BULBS,  PASTEUR'S  APPARATUS, 
MIQUEL'S  BULBS;  DROP-CULTURES. 

Cultivations  in  liquid  media  can  be  carried  on 
in  test-tubes,  but  it  is  more  satisfactory  to  employ 


g2  BACTERIOLOGY. 

special  forms  of  flasks,  bulbs,  U  tubes,  etc.  As 
test-tubes  and  flasks  containing  liquid  media  cannot 
be  inverted,  inoculation  with  a  sterilised  needle  must 
be  carried  out  as  rapidly  as  possible,  with  the  addi- 
tional precaution  of  closed  windows  and  doors. 

Lister's  Flasks. — These  flasks  (p.  31)  were 
especially  introduced  by  Sir  Joseph  Lister  as  a 
means  of  storing  liquid  nutrient  media.  They  are 
so  constructed  that  after  removal  of  a  portion  of 
the  contents,  or  restoring  the  vessel  to  the  vertical 
position,  a  drop  of  liquid  always  remains  in  the 
extremity  of  the  nozzle,  which  prevents  the  regur- 
gitation  of  unfiltered  air. 

Sternberg's  Bulbs. — The  method  of  introducing 
liquid  into  the  bulbs  employed  by  Professor  Stern- 
berg,  and  of  sterilising  and  inoculating  it,  is  as 
follows : — The  bulb  is  heated  slightly  over  the 
flame,  and  the  extremity  of  the  neck,  after  breaking 
off  the  sealed  point,  is  plunged  beneath  the  surface 
of  the  liquid.  As  the  air  cools  the  liquid  is  drawn 
into  the  bulb,  usually  filling  it  to  about  one-third 
of  its  capacity.  The  neck  of  the  flask  is  again 
sealed  up,  and  the  liquid  which  has  been  introduced 
is  sterilised  by  repeatedly  boiling  the  flasks  in  the 
water-bath.  They  should  then  be  placed  in  the 
incubator  for  two  or  three  days,  and  if  the  contents 
remain  transparent  and  free  from  film,  they  may  be 
set  aside  as  stock-bulbs,  to  be  used  when  required. 

To  inoculate  the  liquid  in  the  bulb  the  end  of 
the  neck  is  heated  to  sterilise  the  exterior,  the  bulb 


LIQUID    MEDIA.  93 

is  gently  warmed,  and  the  extremity  of  the  neck 
nipped  off  with  a  pair  of  sterilised  forceps.  The 
open  extremity  is  plunged  into  the  liquid  contain- 
ing the  micro-organism,  a  minute  quantity  enters  the 
tube  and  mingles  with  the  fluid  in  the  bulb,  without 
fear  of  contamination  by  atmospheric  germs.  The 
extremity  of  the  neck  is  once  more  sealed  up  in  the 
flame  of  a  Bunsen  burner. 

Aitken's  Tubes. — These  tubes  are  plugged  and 
sterilised,  and  the  nutrient  medium  introduced  as 
into  ordinary  test-tubes.  Instead  of  withdrawing  the 
cotton  wool  plug  they  are  inoculated  by  means  of 
the  lateral  arm.  The  sealed  extremity  of  the  arm 
is  nipped  off  with  sterilised  forceps,  and  the  in- 
oculating needle  is  carefully  introduced  through 
the  opening  thus  made.  It  is  directed  along  the 
arm  until  it  touches  the  opposite  side  of  the  test- 
tube,  where  it  deposits  the  material  with  which  it 
was  charged.  The  needle  is  withdrawn,  and  the 
end  of  the  lateral  arm  again  sealed  up  in  the  flame; 
the  test-tube  is  then  tilted  until  the  liquid  touches 
the  deposited  material ;  on  restoring  the  tube  to 
the  vertical  the  material  is  washed  down  into  the 
body  of  the  nutrient  liquid. 

Pasteur's  Apparatus.  —  Special  forms  of 
tubes,  bulbs,  and  pipettes  are  employed  by  the 
school  of  Pasteur.  The  tubes  are  provided  with 
lateral  or  with  curved  arms  drawn  out  to  a  fine 
point,  and  with  slender  necks  plugged  with  cotton 
wool.  A  double  form  shaped  like  a  tuning  fork, 


9|  BACTERIOLOGY. 

each  limb  with  a  bent  arm,  is  a  convenient  form  for 
storing  sterilised  bouillon.  The  sealed  end  of  an 
arm  is  nipped  off  with  sterilised  forceps,  the  sterile 
bouillon  aspirated  into  each  limb,  and  the  arm 
again  sealed  in  the  flame ;  a  series  of  such  tubes 
can  be  arranged  upon  a  rack  on  the  working 
table.* 

Bulbs  with  a  vertical  neck  drawn  out  to  a  fine 
point ;  others  with  a  neck  bent  at  an  obtuse  angle 
plugged  with  cotton  wool,  and  a  lateral  curved  arm 
drawn  out  to  a  fine  point,  are  also  employed.  For 
a  description  of  these  various  vessels  and  their 
special  advantages  the  works  of  Pasteur  and 
Duclaux  must  be  consulted. 

Miquel's  Bulbs. — The  tube  a  boule  of  Miquelf 
is  also  a  very  useful  form.  It  consists  of  a  bulb 
of  50  cc.  capacity  blown  in  the  middle  of  a  glass 
tube.  The  part  of  the  tube  above  the  bulb  is  con- 
tracted about  half  way  between  the  bulb  and  its 
extremity,  and  can  either  be  left  quite  straight  or 
can  be  made  to  curve  slightly.  On  either  side  of  the 
contraction  the  tube  is  plugged  with  asbestos.  The 
portion  of  the  tube  below  the  bulb  is  S  shaped,  and 
drawn  out  at  its  extremity  into  a  fine  point.  The 
bulbs  are  charged  with  nutrient  liquid  and  inocu- 
lated by  aspiration,  and  the  point  of  the  S  tube 
sealed  in  the  flame  of  a  Bunsen  burner. 

Drop-Cultures. — This    method    of   cultivation 

*  Duclaux,  Ferments  et  Maladies .     1882. 

t  Miquel,  Les  Organismes  Vivants  de  r Atmosphere.     1883. 


LIQUID    MEDIA.  95 

has  already  been  referred  to  as  a  particularly  in- 
structive one.  It  enables  us  to  study  many  of  the 
changes  which  take  place  during  the  life  history  of 
micro-organisms.  This  is  illustrated,  for  example, 
by  the  anthrax  bacillus,  where  we  can  watch  the 
gradual  growth  of  a  single  bacillus  into  a  long 
filament,  and  the  subsequent  development  of  bright 
oval  spores.  It  is  necessary  carefully  to  observe 
the  minutest  details  to  maintain  the  cultivation  pure. 
An  excavated  slide  is  thoroughly  cleaned,  and 
then  sterilised  by  being  held  with  the  cupped  side 
downwards  in  the  flame  of  the  Bunsen  burner.  A 
ring  of  vaseline  is  painted  round  the  excavation,  and 
the  slide  is  then  placed  under  a  glass  bell.  Mean- 
while a  carefully  cleansed  cover-glass  is  also 
sterilised  by  passing  it  through  the  flame,  and 
should  be  deposited  on  the  plate  of  blackened  glass. 
With  a  sterilised  ose  a  drop  of  sterile  bouillon  is 
transferred  to  the  cover  glass,  and  this  is  inoculated 
by  touching  it  with  a  sterilised  needle  charged  with 
the  material  without  disturbing  the  form  of  the  drop. 
It  is  quite  sufficient  just  to  touch  the  drop  instead 
of  transferring  a  visible  quantity  of  blood,  juice,  or 
growth,  as  the  case  may  be.  The  slide  is  then  in- 
verted and  placed  over  the  cover-glass,  so  that  the 
drop  will  come  exactly  in  the  centre  of  the  excava- 
tion, and  is  gently  pressed  down.  On  turning  the 
slide  over  again  the  cover-glass  adheres,  and  an 
additional  layer  of  vaseline  is  painted  round  the 
edges  of  the  cover-glass  itself.  The  slide  must  be 


96  BACTERIOLOGY. 

labelled,  and,  if  necessary,  placed  in  the  incubator, 
and  the  results  watched  from  time  to  time.  Instead 
of  bouillon  liquid  blood  serum  may  also  be  em- 
ployed in  this  form  of  cultivation.  If  it  be  required 
to  preserve  the  drop  cultivation  as  a  microscopic 
preparation,  the  cover-glass  is  gently  lifted  off  and 
allowed  to  dry.  Any  vaseline  adhering  to  the 
cover-glass  should  be  wiped  off,  and  the  cover-glass 
can  then  be  passed  through  the  flame  and  stained 
in  the  usual  manner. 

Moist  -  Chambers. — Unless  drop-cultures  are 
very  carefully  prepared,  they  are  liable  to  dry  up, 
if  kept  for  examination  for  several  days.  Many 
therefore  prefer  employing  a  moist-chamber.  There 
are  several  different  forms  in  use. 

The  drop-culture  slide  may  be  converted  into  a 
moist-chamber  by  having  a  deep  groove  cut  round 
the  circumference  of  the  concavity.  This  groove  is 
filled  with  sterilised  water  by  means  of  a  pipette. 
A  ring  of  vaseline  is  painted  with  the  earners-hair 
brush  outside  the  groove,  and  the  cover-glass  with 
the  drop-cultivation  is  inverted  and  placed  over  the 
concavity.  This  form  is  very  useful,  as  the  slide 
can  be  easily  cleansed  and  effectually  sterilised  by 
holding  it  in  the  flame  of  the  Bunsen  burner. 

A  very  simple  form  of  moist-chamber  which  may 
be  used  in  some  cases,  but  possesses  the  disadvan- 
tage of  not  admitting  of  sterilisation  by  heat,  may 
be  constructed  as  follows*  : — 

*  Schafer's  Course  of  Practical  Histology.     1877. 


LIQUID    MEDIA.  97 

A  small  piece  of  putty  or  modelling  wax  is  rolled 
into  a  cord  about  two  inches  long  and  J  inch  thick. 
By  uniting  the  ends  a  ring  is  formed,  which  is  placed 
on  the  middle  of  a  clean  glass  slide  (Fig.  31).  A 
drop  of  water  is  placed  in  the  centre  of  the  ring, 
and  the  cell  roofed  in  by  applying  the  cover-glass. 

A  somewhat  similar  cell  in  form,  which  has  the 
advantage  of  permitting  of  thorough  cleansing,  may 


FIG.  31. — METHOD  OF  FORMING  A  SIMPLE  MOIST-CHAMBER. 

be  constructed  by  cementing  a  glass  ring  with  flat 
surfaces  to  an  ordinary  slide.  Vaseline  is  applied 
with  a  earner s-hair  brush  to  the  upper  surface  of 
the  ring,  and  one  or  two  drops  of  water  placed  with 
a  pipette  at  the  bottom  of  the  cell.  The  cover- 
glass,  with  the  preparation,  is  then  inverted  over 
the  cell  and  gently  pressed  down  upon  the  glass 
ring.  The  vaseline  renders  the  cell  air-tight,  and, 
to  a  certain  extent,  fixes  the  cover-glass  to  the  ring. 
Warm  Stages. — To  apply  warmth  while  a  pre- 
paration is  under  continuous  observation  we  must 

7 


98  BACTERIOLOGY. 

either  place  the  microscope  bodily  within  an  in- 
cubator, with  the  eyepiece  protruding-  through  an 
opening,  so  that  we  may  observe  what  is  going  on 
without  moving  the  preparation,  or  we  must  employ 
some  means  of  applying  heat  directly  to  the 
preparation. 

A  simple  warm  stage  may  be  made  of  an  oblong 
copper  plate,  two  inches  long  by  one  inch  wide, 
from  one  side  of  which  a  rod  of  the  same  material 
projects.  The  plate  has  a  round  aperture  in  the 
middle,  half  an  inch  in  diameter,  and  is  fastened  to 


FIG.  32. — SIMPLE  WARM  STAGE. 

an  ordinary  slide  with  sealing  wax.  The  drop  to 
be  examined  is  placed  on  a  large-sized  cover-glass 
and  covered  with  a  smaller  one.  Olive  oil  or 
vaseline  is  painted  round  the  edge  of  the  smaller 
cover-glass  to  prevent  evaporation,  and  the  prepara- 
tion is  placed  over  the  hole  in  the  plate  (Fig.  32). 

The  slide  bearing  the  copper  plate  is  clamped 
to  the  stage  of  the  microscope  (Fig.  33).  The 
flame  of  a  spirit  lamp  is  applied  to  the  extremity 
of  the  rod,  and  the  heat  is  conducted  to  the  plate 
and  thence  transmitted  to  the  specimen.  That  the 


LIQUID    MEDIA. 


99 


temperature  of  the  copper  plate  may  be  approxi- 
mately that  of  the  body,  the  lamp  is  so  adjusted 
that  a  fragment  of  cacao  butter  and  wax  placed 
close  to  the  preparation  is  melted. 

For  more  accurate  observations,  the  apparatus 
shown  in  Fig.  34  may  be  employed.     The  vessel/, 


FIG.  33. — SIMPLE  WARM  STAGE  SHOWN  IN  OPERATION. 

filled  with  water  which  has  been  boiled  to  expel  the 
air,  is  heated  by  means  of  a  gas-flame  at  g.  The 
warmed  water  ascends  the  indiarubber  tube  c  to  the 
brass  box  a.  The  box  is  pierced  by  a  tubular 
aperture  to  admit  light  to  the  object^  and  Iras:  an 
exit  tube  c'9  by  which  tlieTeooled  Vater  ;fitinr  the 


100 


BACTERIOLOGY, 


stage  returns  to  be  reheated  by  the  flame  g.  At  d 
is  a  gas  regulator,  so  that  a  constant  temperature 
at  any  desired  point  can  be  maintained. 


FIG.  34. — SCHAFER'S  WARM  STAGE. 

Another  form  in  which  warm  water  or  steam  can 
be  used  for   heating,   and  by   the  employment  of 


a 

oz 


FIG.  35.— STRICKER'S  WARM  STAGE. 

icea  water  also  used  for  observing  the  effects  of 
Kold^i&shov^in  Fi§x3t5< :  It  consists  of  a  hollow  rect- 
aflgulahf>axv*with"<&  fcfeWtfal  opening  (C)  permitting. 


LIQUID    MEDIA. 


IOI 


the  passage  of  light.  The  water  makes  its  exit  and 
entrance  at  the  side  tubes  a,  a,  and  the  temperature 
is  indicated  by  a  thermometer  in  front. 


FIG.  36.— SECTION  OF  ISRAEL'S  WARMING  APPARATUS  AND 
DROP-CULTURE  SLIDE. 

Israel's  Warming  Apparatus. — It  is  obvious 
that  in  employing  very  high  powers  a  difficulty  will 


FIG.  37. —ISRAEL'S  WARMING  APPARATUS. 

be  presented  by  the   warm  stages  just    described 
owing   to  their  interference  with  the  illumination. 


IO2  BACTERIOLOGY. 

To  overcome  this  an  apparatus  has  been  con- 
structed by  which  the  slide  is  warmed  from  above.* 
The  drop-culture  slides  are  provided  with  a 
shallow  groove  'i  mm.  deep  and  i  mm.  broad,  cut 
round  the  concavity.  Into  this  the  cover-glass  fits, 


FIG.  38.— ISRAEL'S  WARMING  APPARATUS  IN  OPERATION. 

so  that  its  upper  surface  is  flush  with  that  of  the 
slide.    The  heating  apparatus  consists  of  a  flat  disk- 
shaped  box  with  a  central  conical  aperture  (Fig.  36). 
The  entrance  and  exit  pipes  are  fixed  on  at  a 

*  Israel,  Zeitsch.f.  Wiss.  Mikrosc,  ii.,  pp.  459 — 63.     1885. 


LIQUID    MEDIA.  IO3 

right  angle  to  the  side  (Fig.  37).  The  former,  z, 
is  of  metal,  and  the  latter,  a,  of  glass  fitted  with 
a  thermometer,  the  bulb  of  which,  k,  is  contained 
within  the  box.  A  partition,  s,  keeps  up  a  current 
between  the  openings  of  the  pipes,  which  are 
supported  on  a  stand  and  connected  by  tubing 
with  the  hot  water  supply  (Fig.  38). 

A  mixture  of  paraffine  and  vaseline  is  recom- 
mended for  indicating  the  temperature  of  the 
chamber,  and  experience  has  shown  that  if  a  tem- 


FIG.  39. — SIMPLE  GAS  CHAMBER. 

perature  of  37°  C.  be  required  the  temperature  of  the 
water  in  the  box  must  range  between  42°  and  47°  C. 

Gas  Chambers. — To  investigate  the  action  of 
gases  or  vapours  upon  micro-organisms,  a  modi- 
fication of  the  simple  moist  chamber  (Fig.  31), 
may  be  employed  (Fig.  39). 

A  piece  of  glass  tubing  is  first  fixed  to  the  slide 
by  means  of  sealing  wax,  and  the  ring  of  putty  is 
so  placed  as  to  include  the  end  of  this,  leaving 
a  small  interval  at  the  side,  or  a  little  notch  is 
made  in  the  putty  opposite,  so  as  to  afford  an  exit 
for  the  gas  or  vapour  (Fig.  40). 


IO4  BACTERIOLOGY. 

A  more  complicated  apparatus,  combining-  both  a 
warm  stage  and  a  gas  chamber,  is  shown  at  Fig.  41. 
This  consists  of  a  rectangular  piece  of  ebonite  EE 
fixed  to  a  brass  plate  which  rests  on  the  stage  of  the 
microscope.  On  the  upper  surface  of  the  ebonite  is 
another  brass  plate  /,  with  an  aperture  c  leading 
into  a  brass  tube  closed  below  by  a  piece  of  glass. 
To  heat  the  apparatus  the  copper  wire  B  is  placed 


FIG.  40. —GAS  CHAMBER  IN  USE  WITH  APPARATUS  FOR  GENERATING 
CARBONIC  ACID. 

on  the  tube  a,  and  its  extremity  heated  by  the  flame 
of  the  lamp.  The  nearer  the  lamp  to  the  stage 
the  higher  the  temperature,  which  is  indicated  by 
the  thermometer  /.  To  employ  it  as  a  gas  chamber 
the  wire  B  is  laid  aside  and  the  gas  is  conducted 
into  the  chamber  by  the  tube  a  and  escapes  by  the 
tube  a. 

Application    of    Electricity. — To    study    the 


LIQUID    MEDIA. 


effect  of  electricity  we  may  prepare  a  drop -culture 
in  the  moist-chamber  (Fig.  42).  The  cover-glass 
to  be  used  is  provided  with  two  strips  of  tinfoil, 


FIG.  41. — STRICKER'S  COMBINED  GAS  CHAMBER  AND  WARM  STAGE. 

which  are  isolated  from  the  brass  of  the  microscope, 
and  so  arranged  that  a  current  of  electricity  may 
be  passed  through  them  (Fig.  43). 


FIG.  42. — SIMPLE  MOIST-CHAMBER  ADAPTED  FOR  TRANSMISSION 
OF  ELECTRICITY. 


A  much  simpler  plan,  which  may  also  be  employed, 
is  to  take  an  ordinary  glass  slide  and  coat  the  sur- 
face with  gold  size.  The  slide  is  then  pressed 


IO6  BACTERIOLOGY. 

firmly  down  on  gold  leaf  or  tin-foil  and  allowed  to 
dry.    When  dry,  the  metal  is  scraped  away,  leaving 


FIG.  43. — APPARATUS  ARRANGED  FOR  TRANSMITTING  ELECTRICITY. 

two  triangles  with  a  small  interval  between  them,  as 
in  Fig.  44. 


FIG.  44. — SLIDE  WITH  GOLD-LEAF  ELECTRODES. 

The  liquid  containing  the  micro-organisms  is 
placed  between  the  electrodes,  covered  with  a  cover- 
glass,  and  then  subjected  to  the  electric  current. 


CHAPTER  VI. 

EXPERIMENTS  UPON  THE  LIVING  ANIMAL. 

To  carry  out  the  last  of  Koch's  postulates,  and  so 
complete  the  chain  of  evidence  in  favour  of  the 
causal  relation  of  micro-organisms  to  disease,  and 
to  study  the  mode  of  action  of  a  pathogenic  bac- 
terium, it  is  necessary  to  introduce  into  a  living 
animal  a  pure  cultivation  of  the  micro-organism 
in  question.  For  this  purpose  various  animals 
are  employed — such  as  mice,  guinea-pigs,  rabbits, 
pigeons,  and  fowls. 

Inhalation  of  Micro-organisms.  —  The 
animals  may  be  made  to  inhale  an  atmosphere 
impregnated  with  micro-organisms  by  means  of 
a  spray.  In  this  way  Friedlander  succeeded  in 
administering  the  bacteria  of  pneumonia  to 
mice,  and  the  production  of  tuberculosis  by 
experimental  inhalation  has  thrown  light  upon 
the  clinical  records  of  cases  reported  as  instances 
of  the  infectiousness  of  phthisis. 

Administration  with  Food. — A  sheep  fed 
upon  potatoes  which  have  been  the  medium  for 
the  cultivation  of  the  anthrax  bacillus  dies  in  a 


IO8  BACTERIOLOGY. 

few  days.  Similarly,  animals  fed  upon  the  nodules 
of  bovine  tuberculosis  become  tubercular,  and 
even  the  flesh  and  milk  of  tuberculous  animals  will 
occasionally  set  up  tuberculosis. 

Cutaneous  and  Subcutaneous  Inocula- 
tion.— Cutaneous  inoculation  may  be  carried  out 
by  making  a  superficial  wound,  and  inoculating 
it  with  a  sterilised  platinum  needle,  charged 
with  the  micro-organisms  to  be  inoculated.  An- 
other simple  method  is  to  take  a  sterilised  knife, 
infect  the  point  with  the  material  to  be  inoculated, 
and  then  make  a  minute  wound  or  incision.  In 
either  case  a  situation  should  be  selected,  such  as 
the  root  of  the  ear,  which  cannot  be  licked  by 
the  animal  after  the  operation. 

Subcutaneous  inoculation  is  very  simple  and 
effectual,  and  consequently  the  method  most  fre- 
quently employed.  The  animal  selected — for 
example,  a  guinea-pig — is  held  by  an  assistant, 
who  covers  it  with  a  towel,  leaving  only  the  hinder 
extremities  exposed.  By  so  doing,  and  gently- 
laying  it  upon  its  back,  with  its  head  low,  a 
guinea-pig  passes  apparently  into  a  state  of 
hypnotism,  and  the  trivial  operation  can  be  per- 
formed with  little  or  no  movement  on  the  part 
of  the  animal.  From  a  spot  on  the  inner  side 
of  the  thigh  the  hair  is  cut  close  with  a  small 
pair  of  scissors  curved  on  the  flat,  and  the  skin 
must  be  thoroughly  purified  with  sublimate  solu- 
tion. A  small  fold  of  skin  is  then  pinched  up 


EXPERIMENTS    UPON    THE    LIVING    ANIMAL.      IO9 

with  a  pair  of  sterilised  forceps,  and  with  a  pair 
of  sharp  sterilised  scissors,  or  with  a  tenotomy 
knife,  a  minute  incision  is  made.  A  sterilised 
platinum  ose  is  charged  with  the  material  to 
be  inoculated,  and  the  loop  is  gently  inserted 
under  the  skin,  forming  a  small  pocket  in  the 
subcutaneous  tissue.  The  needle  is  then  with- 
drawn, and  the  sides  of  the  wound  gently  pressed 
into  apposition.  In  a  mouse  the  same  process 
is  adopted,  with  the  exception  that  the  root  of  the 
tail  is  the  usual  site  of  the  operation.  In  a  method 
suggested  by  Koch  an  assistant  can  be  dispensed 
with  :  a  glass  bell  reversed  is  placed  as  a  cover 
to  a  wide-mouthed  glass  jar,  in  which  a  mouse 
is  held  by  the  tail  with  a  pair  of  forceps,  while 
the  cover  is  so  placed  over  the  mouth  of  the 
jar  as  to  leave  a  small  interval  near  the  rim 
uncovered.  The  mouse  rests  with  its  head  down- 
wards and  with  its  feet  against  the  inner  wall  of 
the  jar,  and  in  the  interval  between  the  cover 
and  the  rim  the  root  of  the  tail  is  exposed, 
and  must  be  cleansed  and  treated  as  already 
described. 

Special  Operations.  —  In  many  cases  it  is 
absolutely  necessary  to  perform  an  operation  of 
greater  severity.  After  the  administration  of  an 
anaesthetic,  infective  material  may  be  introduced 
into  the  peritoneal  cavity  by  the  performance  of 
abdominal  section,  or  injected  into  the  duodenum 
in  the  manner  employed  in  the  case  of  Koch's 


HO  BACTERIOLOGY. 

comma  bacilli  by  Nicati  and  Rietsch.  In  such 
cases  antiseptic  precautions  must  be  rigidly 
followed,  and  use  made  of  iodoform  and  other 
antiseptic  dressings.  The  disinfection  of  the  skin 
of  the  animal,  of  the  instruments  employed,  and 
of  the  hands  of  the  operator,  are  details  essential 
to  secure  success.  To  inoculate  tubercular  matter, 
sputum  may  be  rubbed  up  with  distilled  water, 
filtered,  and  the  filtrate  injected  into  a  tracheal 
fistula,  or  the  first  steps  of  the  operation  of 
iridectomy  may  be  performed,  and  tubercular 
material  inserted  in  the  anterior  chamber  of  the  eye. 
The  advantage  of  the  latter  method  consists  in  that 
it  enables  the  results  and  changes  to  be  observed 
from  day  to  day.  A  cultivation  of  micro-organisms 
may  also  be  mixed  with  sterilised  water,  and  then 
injected  with  a  syringe  directly  into  the  circulation. 
In  rabbits  this  may  be  performed  without  difficulty 
by  injecting  the  large  vein  at  the  base  of  the  ear 
with  a  Pravaz'  syringe.  Before  every  inoculation 
the  instruments  must  be  sterilised,  as  already  ex- 
plained, by  employing  an  Israel's  case,  and  after 
each  operation  all  instruments  should  be  placed  in 
sublimate  solution,  wiped  dry,  and  sterilised  in  the 
hot-air  steriliser,  before  they  are  put  away.  If  these 
precautions  are  not  observed,  instances  of  accidental 
infection  are  sure  to  occur. 


CHAPTER  VII. 

EXAMTNA  TION  OF  ANIMALS  EXPERIMENTED  UPON 
AND  THE  METHODS  OF  ISOLATING  MICRO- 
ORGANISMS FROM  THE  LIVING  AND  DEAD 
SUBJECT. 

METHOD    OF    DISSECTION    AND    EXAMINATION. 

ALL  animals  that  die  after  an  experimental  inocu- 
lation should  be  examined  immediately  after  death. 
Every  precaution  must  be  taken,  in  conducting 
the  dissection,  to  exclude  extraneous  micro-organ- 
isms, and  all  instruments  employed  must  have  been 
sterilised  in  the  hot-air  steriliser,  or  heated  in  the 
Bunsen  burner.  If  a  mouse,  for  example,  has  died 
after  an  inoculation,  it  should  be  at  once  pinned 
out  by  its  feet  on  a  slab  of  wood  or  in  a  gutta- 
percha  tray,  and  bathed  with  sublimate  solution. 
In  the  same  way,  before  examining  a  dead  rabbit,  a 
stream  of  sublimate  should  be  directed  over  it  to 
lay  the  fur,  which  otherwise  interferes  with  the 
dissection.  The  hair  should  be  cut  away  with 
sterilised  scissors  fro  m>  the  seat  of  inoculation,  which 
is  the  first  part  to  be  examined,  and  any  suppura- 
tion, haemorrhage,  cedema,  or  other  pathological 


I  I  2  BACTERIOLOGY. 

change  should  carefully  be  noted.  From  any  pus  or 
exudation  that  may  be  present,  material  for  inocu- 
lations should  at  once  be  taken,  and  cover-glass- 
preparations  made  for  microscopical  examination. 

To  examine  the  internal  organs  and  to  make 
inoculations  from  the  blood  of  the  heart  or  spleen, 
the  skin  is  cut  through  from  below  upwards  in  the 
median  line  of  the  abdominal  and  thoracic  regions. 
The  abdominal  cavity  is  then  opened,  and  the  walls 
pinned  back  on  either  side  of  the  animal.  Any 
abnormal  appearances  should  be  noted,  and  espe- 
cially the  state  of  the  spleen  should  be  examined, 
by  turning  the  intestines  aside.  After  noting  its 
appearances  it  should  be  removed  with  sterilised 
forceps  and  scissors,  and  deposited  upon  a  sterilised 
glass  slide.  After  washing  it  with  sublimate  solution 
by  means  of  a  camel's  hair  brush  or  strip  of  filter 
paper,  it  should  be  incised  with  sterilised  scissors  ; 
the  pulp  may  be  squeezed  out  from  the  cut  surface, 
and  test-tubes  of  nutrient  gelatine  and  agar-agar 
can  be  inoculated  from  it,  and,  if  necessary,  potato 
and  drop-cultivations  also  established.  Precisely 
the  same  care  must  be  taken  in  examining  lym- 
phatic glands,  tubercles,  or  pathological  nodules; 
any  chance  putrefactive  micro-organisms  on  the 
surface  are  destroyed  by  the  sublimate  solution, 
and  a  section  is  then  made,  and  a  minute  fragment 
snipped  out  of  the  centre  of  the  nodule,  to  be 
examined  or  transferred  to  the  nutrient  medium. 
The  examination  of  the  thorax  is  made  by  cutting 


EXAMINATION  OF  ANIMALS  EXPERIMENTED  UPON.    I  13 

through  the  ribs  on  either  side  of  the  sternum  with 
sterilised  scissors,  and  turning  the  sternum  up 
where  it  will  be  out  of  the  way.  The  pericardium 
is  then  opened,  and  the  right  auricle  or  ventricle 
pierced  with  the  point  of  a  sterilised  scalpel,  and 
inoculations  and  cover-glass-preparations  are  made 
from  the  blood  which  escapes. 

The  lungs  also  require  to  be  especially  studied. 
They  should  be  incised  with  a  sterilised  scalpel, 
and  inoculations  and  cover-glass-preparations  made 
from  the  cut  surface.  It  may  be  necessary  to 
embed  a  piece  of  lung  or  fragment  of  spleen,  so 
that  it  shall  be  free  from  air.  This  may  be  done 
by  isolating  a  fragment  with  the  precautions  just  de- 
scribed and  depositing  it  upon  the  surface  of  a  test- 
tube  of  nutrient  agar-agar.  The  contents  of  another 
tube,  which  have  been  liquefied,  and  allowed  to 
cool  almost  to  the  point  of  gelatinisation,  must  then 
be  poured  over  it.  From  a  potato  a  little  cube 
must  be  cut,  the  tissue  deposited  in  the  trough 
thus  formed,  and  the  cube  replaced.  Blood  may 
also  be  taken  directly  from  a  vein  by  laying  it  bare 
by 'dissection,  making  a  small  section  with  sterilised 
scissors,  and  inserting  an  ose,  the  needle  of  a 
Pravaz'  syringe,  a  capillary  tube,  or  the  extremity 
of  the  capillary  neck  of  a  Sternberg's  bulb.  If  the 
cultivation  is  contaminated  by  the  presence  of 
putrefactive  or  other  micro-organisms  they  must  be 
isolated  subsequently  by  carrying  out  a  series  of 
plate-cultivations. 


I  14  BACTERIOLOGY. 

Having  completed  the  dissection,  the  organs  of 
such  a  small  animal  as  a  mouse  may  be  removed 
en  masse  and  transferred  to  absolute  alcohol  for 
subsequent  examination.  In  other  cases  it  may  be 
only  necessary  to  reserve  portions  of  each  organ. 
In  any  case  it  should  be  remembered  that  with  a 
virulent  micro-organism,  e.g.,  anthrax,  any  remain- 
ing part  of  the  animal  should  be  cremated,  and  the 
hands  and  all  instruments  should  be  thoroughly 
disinfected. 

Isolation  of  Micro-organisms  from  the 
Living  Subject. — Micro-organisms  in  the  living 
subject  may  be  isolated  from  pus  of  abscesses, 
or  other  discharges,  and  from  the  blood  and  tissues. 
Abscesses  should  be  opened,  and  other  operations 
performed,  when  practicable,  with  Listerian  precau- 
tions, and  a  drop  of  the  discharge  taken  up  with  an 
ose  or  capillary  pipette  as  already  explained. 

To  make  a  cultivation  from  the  blood  of  a  living 
person,  the  tip  of  a  finger  must  be  well  washed  with 
soap  and  water  and  bathed  with  strong  sublimate, 
or  i  in  20  carbolic,  solution.  Venous  conges- 
tion is  produced  by  applying  an  elastic  band 
or  ligature  to  the  finger,  which  is  pricked  with  a 
sterilised  sewing  needle.  From  the  drop  of  blood 
which  exudes  the  necessary  inoculations  and  ex- 
aminations can  be  made. 


PART    II. 
GENERAL  BIOLOGY   OF   BACTERIA. 


CHAPTER  VIII. 

GENERAL  MORPHOLOGY  AND  PHYSIOLOGY. 

BACTERIA  may  be  considered  as  minute  vegetable 
cells  destitute  of  nuclei.  They  are  distinguished 
from  animal  cells  by  being  able  to  derive  their 
nitrogen  from  ammonia  compounds,  and  they  differ 
from  the  higher  vegetable  cells  in  being  unable  to 
split  up  carbonic  acid  into  its  elements,  owing  to 
the  absence  of  chlorophyll.  Von  Engelmann  and 
Van  Tieghem  include  among  the  bacteria  certain 
organisms,  named  by  them  Bacttrium  chlorinum, 
Bacterium  vtride,  and  Bacillus  virens,  which  are 
coloured  green  by  this  substance ;  but  further 
researches  are  required  before  any  conclusions  are 
definitely  arrived  at  as  to  the  place  of  these  parti- 
cular organisms  in  the  vegetable  kingdom.  It  is 
quite  possible  that  they  may  be  Algse,  and  they 
will,  therefore,  find  no  place  in  the  classification 
which  will  be  here  adopted. 

Chemical  composition. — For  our  knowledge 
of  the  composition  of  bacteria  we  are  chiefly  in- 
debted to  Nencki.  Their  constituents  are  found  on 


Il8  BACTERIOLOGY. 

analysis  to  vary  slightly,  according  to  whether  the 
bacteria  are  in  zooglcea  or  in  the  active  state.  In 
the  latter  condition  they  are  said  to  consist  of  83*42 
per  cent,  of  water.  In  one  hundred  parts  of  the 
dried  constituents  there  are  the  following : —  . 

A  nitrogenous  body  .                   .  84*20 

Fat     .         .         .         .      t  .         «-  604 

Ash     .         .         .         ...  4-72 

Undetermined  substances     .         .  5-04 

This  nitrogenous  body  is  called  Mycoprotein^  and 
consists  of 

Carbon  -.:  .  .  .  .  52*32 
Hydrogen  .  »  .  .  -7*55 
Nitrogen  .  .  .  •-.-'  14*75 

but  no  sulphur  or  phosphorus. 

The  nitrogenous  body  appears  to  vary  in  different 
species,  for  in  Bacillus  anthracis  a  substance  has 
been  obtained  which  does  not  give  the  reactions 
of  mycoprotein,  and,  therefore,  is  distinguished  as 
anthraxprotein. 

Considering  bacteria  as  cells,  we  may  speak  of 
the  cell-wall  and  the  cell-contents. 

Cell-wall. — The  cell-wall  consists  of  cellulose, 
or  according  to  Nencki  in  the  putrefactive  bacteria 
of  mycoprotein.  It  may  be  demonstrated  by  the 
action  of  iodine,  which  contracts  the  protoplasmic 
contents,  and  renders  the  cell-wall  visible.  The 
author  has  taken  advantage  of  the  action  of  iodine 
to  differentiate  by  staining  the  sheath  of  the  Bacillus 


GENERAL    MORPHOLOGY    AND    PHYSIOLOGY.       IIQ 

anthracis  from  its  contents.  If  we  stain  cover-glass 
preparations  of  this  bacillus  by  the  method  of 
Gram,  we  get  the  following  results.  By  the  first 
solution  the  rods  are  uniformly  stained  blue  ;  by 
subjecting  them  to  the  iodine  solution,  the  proto- 
plasmic contents  are  contracted, 
while  the  next  solution,  alcohol, 
decolorises  the  sheath,  which  may 
be  then  stained  in  contrast  with 


The     cell-wall     may    be     either 
pliable  or  rigid.     Pliability  is  ob-  FlG-45- 

served  in  the  long  filaments,  which 
are   endowed   with    a   slow   vermi-     THRACIS,     DOUBLE 

,    .,  .      .   ,.  STAINED  WITH  GEN  - 

cular     movement,     while     rigidity     TIAN  VIOLET  AND 
accounts    for    the    maintenance   of     EOSIN\  ^heS1heat!j 

was  stained  pink,  and 

the   characteristic  form    of  several     the     ceil  -  contents 

i  'MI  blue,     x  1200. 

species,  such  as  spirilla. 

Cell  -  contents.  —  The  cell  -  protoplasm  yields 
mycoprotein.  In  some  it  is  homogeneous,  and 
in  others  granular.  The  action  of  the  aniline  dyes 
indicates  a  close  relation  to  nuclear  protoplasm, 
though  all  nuclear  stains  are  not  suitable  for 
bacteria.  In  some  cases  also,  the  bacteria  remain 
stained  under  the  influence  of  a  reagent,  which 
removes  the  colour  from  nuclei.  The  power  of  fixing 
the  stain  is  not  always  present,  and  indicates  a 
difference  in  the  protoplasm  of  different  species. 
Thus  in  staining  phthisical  sputum,  the  nitric  acid 
removes  the  stain  from  all  bacteria  and  bacilli 


1 2O  BACTERIOLOGY. 

present,  with  the  exception  of  the  tubercle  bacillus. 
This  difference  in  the  protoplasm  of  different 
species  is  also  illustrated  by  the  necessit}'  in  many 
cases  of  using  special  processes,  owing  to  the 
ordinary  methods  being  unsatisfactory  or  not  pro- 
ducing any  result. 

The  protoplasm  of  some  bacteria  contains  starch 
granules ;  thus  Closlridium  butyricum  gives  the 
starch  reaction  with  iodine.  Sulphur  granules  are 
present  in  some  species  of  Beggiatoa  which  thrive 
in  sulphur  springs.  The  colouring  matter  of  the 
pigment  bacteria  is  probably  external  to  the  cell 
as  a  rule ;  for  example,  in  Bacterium  prodigiosum 
the  pigment  granules  are  distinctly  between  the 
cells ;  on  the  other  hand,  in  Beggiatoa  roseo- 
persicina,.  or  the  peach-coloured  bacterium,  the 
special  pigment  bacterio-purpunn  appears  to  be 
dissolved  in  the  cell  protoplasm.  In  Bacillus 
pyocyaneus  the  pigment  is  certainly  not  localised 
entirely  in  the  cell ;  for  it  becomes  rapidly  diffused 
in  the  surrounding  medium,  considerably  beyond 
the  confines  of  the  growth  itself. 

Gelatinous  envelope. — In  several  species,  either 
as  a  result  of  a  secretion  from  the  cell,  or  of  the 
absorption  of  moisture  and  swelling  up  of  the  outer 
layer  of  the  cell-wall,  a  mucinous  or  gelatinous 
envelope  develops  around  them.  This  envelope 
may  form  a  capsule,  such  as  we  meet  with  in 
certain  bacteria  found  in  the  rusty  sputum  of 
pneumonia,  and  in  Micrococcus  tetragonus  ;  or  it  may 


GENERAL    MORPHOLOGY   AND    PHYSIOLOGY.       121 

occur  as  a  continuous  sheath  around  a  chain 
of  bacteria,  which  by  its  disappearance  sets  the 
individual  links  free.  The  capsule  is  soluble  in 
water,  and  under  some  circumstances  is  difficult  to 
demonstrate.  In  the  pneumo-coccus  of  Friedlander 
the  capsule  disappears  on  cultivation,  but  reappears 
in  preparations  made  from  an  inoculated  animal. 
In  the  pleuritic  fluid  of  a  mouse  these  cocci  are 
often  found  with  a  strikingly  well-marked  capsule, 
and  in  other  capsuled  cocci  the  extent  of  the 
envelope  has  been  observed  to  vary  considerably 
in  the  same  species  of  bacterium. 

When  this  gelatinous  material  forms  a  matrix,  in 
which  numbers  of  bacteria  are  congregated  in  an 
irregular  mass,  we  have  what  is  termed  a  zoogloea. 
Thezooglcean  stage  is  a  resting  stage,  often  preceded 
or  followed  by  a  motile  stage.  Thus  bacteria  may 
be  present  in  a  solution  in  an  active  state,  and  after 
a  time  a  scum  or  pellicle  forms  on  the  surface  of  the 
liquid,  which  consists  of  zooglcea.  At  the  edges  of 
the  zooglcea  individuals  may  be  seen  again  to 
become  motile,  detaching  themselves  from  the 
edges  of  the  mass,  and  swimming  off  in  the 
surrounding  fluid. 

The  same  may  be  observed  sometimes  in  culti- 
vations started  in  nutrient  gelatine.  The  inoculated 
bacteria  grow  and  multiply,  and  liquefy  the  gelatine, 
and  after  a  time  a  zooglcean  film  appears  on  the 
surface  of  the  liquefied  layer.  On  potatoes  the 
appearances  are  very  varied.  In  a  bacillus  which 


122 


BACTERIOLOGY. 


readily  develops  on  unsterilised  potatoes,  the  zoo* 
glcea  may  spread  over  the  cut  surface,  forming  a 
pellicle  which  can  be  raised  en  masse  like  a  delicate 
veil.  Another  bacillus  forms  a  zooglcea,  consist- 
ing of  a  tenacious  layer  which  can  be  drawn  out 
in  long  stringy  threads.  In  Ascococcus  Billrothii  the 
gelatinous  envelope  develops  to  such  an  enormous 
extent  that  it  forms  the  characteristic  feature  of  the 
species  (Fig.  46). 


FIG.  46.— Ascococcus  BILLROTHII,  X  65.    [After  Cohn.] 

Form. — The  individual  cells  vary  in  form,  and 
may  either  remain  isolated  or  attached  to  each 
other.  Round  cells  and  egg-shaped  cells  are  called 
cocci.  The  spherical  form  is  the  most  common, 
but  cocci  are  occasionally  exclusively  ovoid,  as  in 
Streptococcus  bombycis.  The  giant  cocci  of  some 
species  are  spoken  of  as  megacocci,  to  distinguish 
them  from  the  ordinary  cocci,  such  as  micrococci. 
The  fission  by  which  the  cocci  increase  may  take 
place  in  one  direction  only,  and  if  the  two  resulting 


GENERAL    MORPHOLOGY   AND    PHYSIOLOGY.       123 

cells  remain  attached  to  each  other,  they  form 
a  diplococcus.  If  these  two  cells  again  divide,  and 
the  resulting  cells  remain  linked  together,  we  get 
a  chain  or  rosary,  or  streptococcus  (Figs.  47,  48,  49). 


FIG.  47. — STREPTOCOCCUS  AND          FIG.  48.— STREPTOCOCCUS  IN  THE 
SARCINACOCCUS     FROM     A  BLOOD  OF  A  RABBIT,  x  1200. 

DROP-CULTIVATION,  x  1200. 

These  chains  may  consist  of  a  few — four  or  five — 
individuals  linked  together,  or  of  a  far  greater 
number,  in  which  case  the  chains  are  generally 
curved  or  twisted.  If  the  division  occur  in  two 


FIG.  49.— STREPTOCOCCUS  OF  PROGRESSIVE  TISSUE  NECROSIS  IN  MICE. 
[After  Koch.] 

directions,  so  that  four  cocci  result,  a  tetrad  or  meris- 
mopedia  is  formed.  If  the  division  occur  in  three 
directions,  one  coccus  divides  into  eight,  and  we 
get  a  packet  form  or  sarcinacoccus.  Immediately 
after  division  the  daughter  cells  are  not  perfectly 


1 24  BACTERIOLOGY. 

circular,  but  are  flattened  or  facetted  where  they 
are  opposite  to  each  other.  They  gradually 
become  rounded  off,  and  each  daughter  cell  is  then 
ready  to  divide  in  its  turn.  In  other  cases  the 
cocci  after  division  only  form  irregular  heaps  or 
collections  like  bunches  of  grapes.  This  form  is 
sometimes  distinguished  as  staphylococcus^  but  it 
cannot  be  considered  an  important  feature.  Where 
we  find  irregular  masses  or  balls  embedded  in  a 
copious  gelatinous  matrix,  the  extent  of  the  latter 
affords  a  characteristic  condition  described  as 
ascococcus. 

Another  type  is  the  rod,  characteristic  of  bac- 
terium and  bacillus.  The  rods  may  vary  con- 
siderably in  length.  The  very  short  rods  with 
rounded  ends  are  very  difficult  to  distinguish  from 
the  oval  cocci,  but  differ  in  that  a  rod,  however 
short  it  may  be,  must  have  at  least  two  sides 
parallel.  The  vibrio  or  bent  rod  may  be  considered 
as  the  connecting  link  between  the  rods  and  the 
corkscrew  forms  or  spirilla.  Lastly  we  have  the 
filamentous  forms,  which  may  be  straight,  leptothrix, 
or  wavy,  spirochceta  (Fig.  50),  or  the  wavy  thread 
may  be  looped  and  entwined  on  itself,  spirulina 
(Plate  L,  Fig.  37). 

By  involution  forms  we  signify  certain  irregular 
shapes  which  result  especially  in  exhausted  culti- 
vations. They  are  peculiar,  oval,  pear-shaped,  or 
irregular  enlargements  (Plate  I,  Figs.  31  to  36). 

Movement. — Many  bacteria  are  devoid  of  move- 


GENERAL    MORPHOLOGY   AND    PHYSIOLOGY.       125 

ment  throughout  the  whole  of  their  life  history. 
Others,  during  certain  stages  of  their  life  cycle,  and 
possibly  some  forms  always,  are  endowed  with 
locomotive  power.  The  character  of  the  movement 
is  very  varied,  and  ranges  from  a  slow  undulatory 
motion  to  one  of  extreme  rapidity.  Many  appear 
to  progress  in  a  definite  direction.  Others  move 
continuously,  first  in  one  direction  and  then  in 
another,  and  others  again  seem  to  hesitate  before 
altering  their  course.  They  may  either  glide  along 
smoothly  or  progress  with  a  tremulous  action. 
They  appear  to  be  able  to  avoid  obstacles,  and  to 


- 

FIG.  50.— SPIROCH^ETA  FROM  SEWAGE  WATER,  x  1200. 

set  themselves  free  from  objects  with  which  they 
have  accidentally  come  into  contact.  Vibrios  have 
a  peculiar  serpentine  movement,  but  other  forms, 
such  as  the  commonly-known  Bacterium  termo  and 
segments  of  spirilla,  such  as  comma-bacilli,  revolve 
around  their  long  axis  as  well  as  make  distinct 
progression.  The  comlpete  spirilla  are  charac- 
terised by  the  familiar  corkscrew  movement. 
With  regard  to  cocci  there  is  some  doubt  as  to 
whether  they  are  endowed  with  independent  move- 
ment; any  quivering  or  oscillation  is  generally 
regarded  as  only  brownian  or  molecular.  In  some 


126  BACTERIOLOGY. 

straight  thread-forms,  which  are  motile,  the  move- 
ment is  very  slow  and  vermicular  in  character,  but 
in  wavy  threads,  such  as  the  Spiroch&U  plicatilis^ 
there  is  not  only  an  undulatory  motion,  with  rapid 
progression  across  the  field  of  the  microscope,  but 
if  they  are  confined  by  more  or  less  debris,  they 
give  very  peculiar  and  characteristic  spasmodic 
movements. 

The  rod- forms  of  Proteus  vulgaris  exhibit  very 
extraordinary  movements  on  the  surface  of  solid 
nutrient  gelatine.  Groups  of  rods  may  be  observed 
to  pass  each  other  in  opposite  directions.  Single 
individuals  meet  and  progress  side  by  side,  or  one 
or  more  individuals  may  part  from  a  group  and 
glide  away  independently.  Occasionally  a  number 
of  rods  progress  in  single  file.  It  is,  however, 
difficult  to  believe  that  these  movements  can  occur 
on  a  solid  surface.  The  author  is  inclined  to 
believe  that  there  is  an  almost  inappreciable  layer 
of  liquid  on  the  surface  of  the  gelatine,  which  is 
expressed  after  the  gelatine  sets.  In  tubes  of 
nutrient  agar-agar  gelatinised  obliquely  and  then 
kept  upright  the  liquid  so  expressed  collects  at  the 
bottom  of  the  sloping  surface. 

What  the  means  are  by  which  bacteria  are  en- 
dowed with  the  power  of  spontaneous  movement 
and  of  progression  may  still  be  said  to  be  unsettled. 
The  author  has  watched  the  movement  of  long 
slender  threads  in  sewage-contaminated  water, 
which  could  only  be  explained  by  the  inherent 


GENERAL    MORPHOLOGY    AND   PHYSIOLOGY.        127 


contractility  of  the  protoplasmic  contents ;  for  if 
any  drawing  or  propelling  organ  existed  in 
proportion  to  the  length  of  the  organism,  it  would 
probably  have  been  visible.  But  in  many  cases  the 
organism  is  undoubtedly  provided  with  a  vibratile 


9  .%° 


FIG.  51. 

I.  Coccus  with  flagellum.  2.  Similar  coccus  dividing  with  two  flagella. 
3.  Colony  of  flagellated  macrococci  of  Beggiatoa  roseopersicina.  4.  Short 
rod  from  the  same  Beggiatoa  with  flagella  [all  after  Zopf  ].  5.  Bacillus 
with  flagella  [from  a  photograph  by  Koch].  6.  Bacillus  subtilis  [after 
Brefeld].  7,  8.  Short  rod-forms  of  Beggiatoa  roseopersicina  with  one 
flagellum  [after  Zopf].  9.  Very  long  rod  of  the  same,  with  flagellum  at 
both  ends  [after  Warming].  10.  Vibrio,  with  double  flagellum  at  each 
end  [after  Warming].  II.  Vibrio,  with  flagella  [from  a  photograph  by  the 
author].  12.  Spirillum  with  flagella  [after  a  photograph  by  Koch]. 
13.  Spirillum  with  flagella  [after  Zopf].  14.  Spirillum  with  double  flagella 
[after  Zopt].  15.  Beggiatoa  roseopersicina  with  a  triple  flagellum  at  one 
end  ;  and  16.  with  a  double  flagellum  at  both  ends  [after  Warming]. 

lash  or  flagellum  at  one  end,  or  with  one  or  more 
at  both  ends  (Fig.  51). 


128  BACTERIOLOGY. 

Some  observers  believe   that   the    movement  of 
cocci  is  due  to  the  existence  of  a  flagellum.     In 
Bacterium  termo  the  existence  of  a  lash  at  either  end 
was  first  determined  by  the  researches  of  Dallinger 
and  Drysdale.     In  motile  bacilli,  such  as  the  hay 
bacillus  and  Bacillus  ulna,  and  in  vibrios  and  spirilla, 
the  flagella  can   be  readily  recognised   by  expert 
microscopists   with    the    employment   of  the   best 
lenses,  and,  what  is  of  equal  importance,  proper 
illumination.     They  are  objects  of  extreme  delicacy, 
and  tenuity,  and  in  stained   preparations    may   be 
absent  from  retraction  or  injury.      Koch  succeeded 
in  photographing  them  after  staining  with  logwood, 
which  turned  them  a  brown  colour.     They  may  also 
be  stained  with  the  aniline  dyes,  for  the  author  has 
observed  them   in  vibrios  in   preparations  stained 
with  gentian  violet,  from  which  also  they  have  been 
photographed,  in  spite  of  the  violet  colour,  by  the 
use  of  isochromatic  dry  plates. 

It  is  not  certain  whether  the  flagella  are  exten- 
sions of  the  cell-wall,  or  derived  from  the  internal 
protoplasm.  Van  Tieghem  holds  the  first  view,  and 
does  not  regard  them  as  motile  organs  at  all.  Zopf, 
on  the  other  hand,  adheres  to  the  second  view,  and 
moreover  believes  that  they  can  be  retracted  within 
the  cell-wall. 

Reproduction. — Bacteria  multiply  by  fission, 
and  by  processes  which  may  be  considered  as 
representing  fructification.  The  bacteria  exhibiting 
the  latter  processes  have  been  divided  into  two 


GENERAL    MORPHOLOGY    AND    PHYSIOLOGY.       I2Q 

groups,  distinguished  by  the  formation  of  endo- 
spores  in  the  one,  and  of  arthrospores  in  the  other. 
In  the  process  of  fission  the  cell  first  increases  in 
size,  and  a  transverse  septum  forms  from  the  cell-wall, 
dividing  the  internal  protoplasm  into  two  equal 
parts ;  these  may  separate  and  lead  an  independent 
existence,  or  remain  linked  together.  In  chains  of 


P 


FIG.  52. — BACILLUS  MEGATERIUM. 

•         a.  A  chain  of  rods.  X  250.    The  rest  X  600. 
b.  Two  active  rods. 

d  to/.  Successive  stages  of  spore-formation. 
h  and  /.   Successive  stages  of  germination. 

[After  DeBary.] 

cocci  the  individual  cells  are  easily  visible  and 
distinct,  but  in  the  thread-forms  resulting  from  the 
linking  together  of  rods,  as  in  the  anthrax  bacillus, 
the  composition  of  the  thread  is  only  demonstrated 
by  the  action  of  reagents. 

Endospore  formation  may  be  conveniently  studied 
in  Bacillus  anthracis,  Bacillus  megaterium>  or  Bacil- 
lus subtilis.  The  protoplasm  becomes  granular, 

9 


130 


BACTERIOLOGY. 


and  at  certain  points  in  the  thread  a  speck  ap- 
pears, which  gradually  enlarges  and  develops  into 
a  circular  or  egg-shaped,  sharply  defined,  highly 
refractive  body.  The  spore  grows  at  the  expense 
of  the  protoplasm  of  the  cell,  which  in  time,  to- 
gether with  the  cell-wall,  entirely  disappears,  and 
the  spore  is  set  free.  These  phenomena  are  best 
seen  in  an  immotile  bacillus  in  a  drop-cultivation 


FIG,  53.— CLOSTRIDIUM  BUTYRICUM,  x  1020. 

B.  Stages  of  spore-formation. 

C.  Stages  of  germination. 

[After  Prazmowski.] 

on  a  warm  stage,  the  whole  process  may  then 
be  observed  continuously  from  beginning  to  end. 
Spores  may  form  in  each  link  of  the  thread,  so 
that  a  regular  row  results,  or  they  may  occur  at 
irregular  intervals.  Spore-formation  also  occurs  in 
free  rods  in  the  centre  or  at  one  end.  Occasionally 
a  spore  develops  at  the  extreme  end,  giving  a  bacillus 
.the  appearance  of  a  drum-stick.  The  spore  may  be 


GENERAL    MORPHOLOGY    AND    PHYSIOLOGY.        13! 

considerably  wider,  but  is  never  longer  than  the 
parent  cell. 

Arthro-spore  formation  is  illustrated  in  Leuco- 
nostoc  mesenteroides.  Certain  elements  in  the  chain 
of  cocci,  apparently  not  differing  from  the  rest, 


FIG.  54. — A  THREAD  OF  BACILLUS  ANTHRACIS  WITH  SPORES,  IN  A 
DROP-CULTIVATION,  x  1400. 

become  larger,  with  tougher  walls,  and  more  refrac- 
tive (Fig.  55).  The  remaining  cells  die,  and  these 
cells  having  acquired  the  properties  of  spores  are 
set  free,  and  can  reproduce  a  new  growth  in  any 


FIG.  55. — LEUCONOSTOC  MESENTEROIDES;  COCCI-CHAINS  WITH 
ARTHROSPORES  (after  Van  Tieghem  and  Cienkowski). 

fresh  nourishing  soil.  That  this  occurs  in  all  species 
which  do  not  form  endospores  is  at  present  only 
a  supposition. 

Spores  are  invested  by  a  thick  membrane,  which 
is  believed  to  consist  of  two  layers.  To  this  they 
probably  owe  the  property  they  possess  of  retaining 
vitality  when  desiccated,  and  of  offering  a  greater 


132  BACTERIOLOGY. 

resistance  to  the  action  of  chemical  reagents  and 
heat  than  the  parent  cells. 

Spore-formation  has  been  regarded  by  some  as 
occurring  when  the  nourishing  soil  is  exhausted, 
thus  providing  for  the  perpetuation  of  the  species. 
For  instance,  anthrax  bacilli  do  not  form  spores  in 
the  living  body,  but  when  the  animal  dies  it  has 
been  stated  that  development  of  spores  takes  place, 
and  hence  the  danger  of  contaminating  the  soil  if 
the  body  is  disposed  of  by  burial.  Klein,  however, 
has  pointed  out  that  if  mice  and  guinea-pigs  which 
have  died  of  anthrax  are  kept  unopened,  the  bacilli 
simply  degenerate  and  ultimately  disappear.  Thus 
there  is  good  reason  for  believing  that  spore-forma- 
tion is  not  due  to  exhaustion  of  the  pabulum,  but 
probably  free  access  to  oxygen  constitutes  an  im- 
portant factor  in  inducing  this  condition.  If  we 
inoculate  a  potato  with  anthrax,  copious  spore-for- 
mation occurs,  though  we  cannot  consider  that  the 
nourishing  soil  has  been  exhausted.  But  we  have 
in  this  case  the  surface  of  the  potato  freely  exposed 
to  the  air  in  the  damp-chamber.  In  the  same  way, 
in  cultivations  on  agar-agar  solidified  obliquely,  so 
as  to  get  a  large  surface,  spore- formation  readily 
takes  place.  Contamination  of  a  burial-ground 
must  result,  therefore,  from  bodies  in  which  a  post- 
mortem examination  has  been  made,  by  which  the 
blood  and  organs  have  been  freely  exposed  to 
the  air,  or  from  animals  which  have  not  been 
examined,  owing  to  their  hides  being  soiled  with 


GENERAL   MORPHOLOGY   AND    PHYSIOLOGY.       133 


excretions,  and  with  blood  which  issues  from  the 
mouth  and  nostrils  before  death. 

When  spores  are  introduced  into  a  suitable 
medium  at  a  favourable  temperature,  they  develop 
again  into  rods.  The  spore  loses  its  sharp  con- 
tour, and,  at  one  pole  or  on  one  side,  a  pale  process 
bursts  through  the  membrane,  gradually  growing 
into  a  rod  from  which  the  empty  capsule  is 
thrown  off  (Figs.  52  and  53). 

Spores  differ  from  the  parent  cells  in  their  be- 


FIG.  56.— SPORES  OF  BACILLUS  AN- 
THRACIS, STAINED  WITH  GENTIAN 

VIOLET,  AFTER  PASSING  THE 
COVER-GLASS  TWELVE  TIMES 
THROUGH  THE  FLAME,  X  I2OO. 


%   [  ;aJ 


FIG.  57.—  SPORE-BEARING  THREADS 
OF  BACILLUS  ANTHRACIS, 
DOUBLE-STAINED  WITH  FUCH- 
SINE  AND  METHYLENE  BLUE, 
X  1200. 


haviour  to  staining  reagents.  Like  them,  they  can 
be  stained  with  aniline  dyes,  but  not  by  the 
ordinary  processes.  They  require  to  be  specially 
treated.  This  is  probably  due  to  the  tough  capsule, 
which  must  first  be  altered  or  softened  by  heat  or 
strong  acid,  until  it  allows  the  stain  to  penetrate. 

Once  stained,  they  again  differ  from  the  parent 
cells  in  resisting  decolorisation  ;  this  fact  is  taken 
advantage  of  to  double-stain  spore- bearing  bacilli. 

In  staining  micro-organisms,  the  protoplasm  is 


134  BACTERIOLOGY. 

sometimes  broken  up  into  irregular  segments  or 
granules,  as  in  many  spirilla,  and  we  may  perhaps 
add  the  bacilli  of  tuberculosis  and  leprosy.  The 
beaded  appearance  of  the  tubercle  bacillus  is  well 
known.  Some  observers  have  regarded  the  beads, 
others  the  bright  spaces  between  'them,  as  spores. 
But  spores  in  unstained  preparations  appear  as 
glistening  bodies  with  sharp  contour,  and  do  not 
stain  at  all,  or  very  little,  by  the  ordinary  processes. 
It  appears,  therefore,  very  doubtful  whether  either 
the  clear  spaces  or  the  beads  are  spores,  espe- 
cially as  the  tubercle  bacillus,  when  unstained, 
is  a  slightly  curved  hyaline  rod,  without  any  dif- 
ferentiation into  granules.  These  considerations 
led  the  author  to  stain  and  examine  tubercular 
sputum  from  various  sources  under  careful 
illumination,  and  with  such  lenses  as  Powell  and 
Lealand's  -^  in.  Horn.  imm.  The  tubercle  bacillus 
may  then  be  frequently  seen  to  consist  of  a  very 
delicate  sheath,  holding  together  a  number  of 
deeply-stained  granules,  for  the  most  part  round 
or  cylindrical,  with  irregular  contour,  and  differing 
considerably  in  size,  while  the  light  interspaces 
are  seen  to  vary  in  form  according  to  the  shape 
of  the  granules.  In  some  preparations  more  dis- 
tinct, and  clearly  ovoid,  granules  may  be  observed 
which  are  sometimes  terminal.  They  can  be  readily 
demonstrated  by  taking  a  photograph  with  a  -£$  in. 
Horn,  imm.,  and  subsequently  enlarging  the  nega- 
tive to  from  2,500  (Fig.  58)  to  6,000  diameters. 


GENERAL    MORPHOLOGY   AND    PHYSIOLOGY.        135 

It  is  not  impossible  that  these  ovoid  granules  are 

spores,     which,     in     their 

behaviour  towards  staining 

reagents,     thus    form     an 

exception    to    the   general 

rule.       But   there   can    be    ^ 

little  doubt  that  a  tubercle          ^^. 

bacillus   consists,    for    the  * 

most  part,  of  a  very  delicate 

FIG.   58.— TUBERCLE  BACILLI   IN 

sheath,   with    protoplasmic      SPUTUM,  x  2500  (from  photo- 


contents  which  have  a  great 

tendency  to  be  broken  up  or  coagulated  into  little 

—  ^x         segments   or    roundish    granules,    owing 

(  ^V     possibly  to  the  treatment  they  are  sub- 

jected   to    in    making    a    microscopical 

FIG.  59.  .  -T-I  •        i_  j 

LEPROSY  BA-  Preparation.        Inis,   however,    does  not 
CILLI,  FROM  always  occur,  for  the  bacilli  at  times  are 

A    SECTION  111  .        ,    .        ,     . 

OF     SKIN,  not  beaded,  but  are  stained  in  their  en- 
x  I200<       tirety.       In  the  leprosy  bacilli  a  similar 

appearance  occurs.      In  stained  sections  the  rods 

have  a  beaded  appearance,  but  the 

intervals  between   the  granules  are 

sometimes  very  long,  and  occasion- 

ally  the  protoplasm  appears  to  have 

collected  only  at  the  extreme  ends  FIG.  60. 

of  the  rod  (Fig.  59).     Very  probably 


the  appearances  in  the  case  of  the      OF  A   GLANDERS 

•11  r      i       j  /T-         r    \  j        NODULE,  x  1200. 

bacillus  of  glanders  (rig.  oo),  and 

the  bacterium  of  chicken-cholera  (Figs.  61  and  62) 

may  be  similarly  explained. 


136 


BACTERIOLOGY. 


The  fact  that  tubercular  sputum  preserves  its 
virulence  for  several  months,  even  after  desicca- 
tion, has  been  attributed  to  the 
formation  of  spores,  and  Babes 
has  drawn  attention  to  ovoid 
grains  in  old  cultivations  of  the 
bacilli,  which  he  succeeded  in 
staining  red,  while  the  bacilli 
were  stained  blue. 

BACTERIUM   OF   CHICKEN-          j       faj       definition    of    spirilla 
CHOLERA,   FROM   BLOOD 

OF  INFECTED  HEN,  x    Zopf  gives  the  spore-formation 

as    absent    or    unknown.      In 

comma-bacilli  in  sewage  water,  the  author  has  often 

noted   appearances   very   suggestive   of   refractive 


FIG.  61. 


FIG.  63. 

COMMA  BACILLI  IN  SEWAGE 
WATER,  STAINED  WITH 
GENTIAN  VIOLET,  x  1200. 


I 

i 

FIG.  62. 

BACTERIUM  OF  CHICKEN-CHOLERA, 
FROM  MUSCLE  JUICE  OF  AN  IN- 
FECTED HEN,  x  2500  [from  a 
photograph]. 

spores  (Fig.  63).  The  same  also  may  be  observed 
in  vibrios,  differing  by  their  regular  contour  from 
the  irregular  spaces  occasionally  observed  in  stained 
preparations  (Figs.  64  and  65).  They  are  possibly 
only  vacuoles. 

Respiration  and  nutrition. — Like  all  a-chlo- 
rophyllous  vegetables,  bacteria  require  for  their 
nutrition  oxygen,  nitrogen,  carbon,  water,  and 
certain  mineral  salts.  Many  require  free  access  to 
oxygen,  others  can  derive  it  from  the  oxidised 


GENERAL    MORPHOLOGY   AND    PHYSIOLOGY.       137 

compounds  in  the  medium  in  which  they  grow. 
Pasteur  divided  bacteria  into  two  great  classes, 
the  aerobic  and  anaerobic ;  and  considered  that  the 
latter  not  only  had  no  need  for  oxygen,  but  that  its 
presence  was  actually  deleterious.  Though  this 
view  must  be  considerably  modified,  the  terms  are 
convenient,  and  are  still  retained.  They  are  well 
illustrated  by  the  bacillus  of  anthrax,  and  the 
bacillus  of  malignant  oedema  ;  and  a  simple  plan  of 
demonstration  has  been  employed  by  the  author. 
A  fragment  of  tissue  from  the  spleen,  for  example, 


FIG.  64.  FIG.  65. 

VIBRIOS  IN  WATER  CONTAMINATED  SPIRILLUM  UNDULA, 

WITH  SEWAGE,  x  1200.  x  1200. 

known  to  contain  anthrax  bacilli,  is  deposited  with 
a  sterilised  inoculating  needle,  with  the  necessary 
precautions,  on  the  surface  of  nutrient  agar-agar 
in  a  test-tube ;  another  tube  of  nutrient  agar-agar 
is  liquefied,  and  when  cooled  down  almost  to  the 
point  of  gelatinisation,  a  part  is  poured  into  the 
first  tube,  so  that  when  it  sets  the  piece  of  tissue 
is  completely  embedded.  A  piece  of  tissue  from 
an  animal  suffering  from  malignant  oedema  is 
treated  in  the  same  way,  and  the  tubes  are 
placed  in  the  incubator.  If  then  we  examine 


138  BACTERIOLOGY. 

them  after  two  or  three  days,  we  shall  find  no 
change  in  the  anthrax  tube  ;  the  bacillus  being 
eminently  aerobic,  no  growth  whatever  has  oc- 
curred. In  the  tube  containing  the  bacilli  of 
malignant  oedema  there  will  be  a  more  or  less 
characteristic  cultivation. 

The  nitrogen  which  is  essential  for  building  up 
their  protoplasm  can  be  obtained  either  from 
albumins,  or  from  ammonia  and  its  derivatives. 
That  the  albumins  can  be  dispensed  with  was 
shown  by  Pasteur,  who  employed  an  artificial 
nourishing  solution  built  upon  a  formula  repre- 
senting the  essential  food  constituents  (p.  91). 

Carbon  is  derived  from  such  substances  as  cane 
sugar,  milk  sugar,  and  glycerine,  and,  in  some 
cases,  by  the  splitting  up  of  complex  proteid 
bodies. 

Water  is  essential  for  their  growth,  but  depriva- 
tion of  water  does  not  kill  all  bacteria.  Desicca- 
tion on  potato  is  employed  for  preserving  some 
micro-organisms,  as  a  new  growth  can  be  started, 
when  required,  by  transferring  some  of  the  dried 
potato  to  fresh  nourishing  ground.  Comma-bacilli, 
on  the  other  hand,  are  destroyed  by  drying.  Sugar, 
by  abstracting  water,  prevents  the  development  of 
micro-organisms  in  preserves. 

Mineral  or  inorganic  substances,  such  as  com- 
pounds of  sodium  and  potassium,  and  different 
phosphates  and  sulphates,  are  necessary  in  small 
proportions. 


GENERAL    MORPHOLOGY    AND    PHYSIOLOGY.       139 

Circumstances      affecting     their    growth. 

Nature  of  the  Soil.  —  Though  we  know  the 
elements  necessary,  we  are,  nevertheless,  as  yet 
unable  to  provide  a  pabulum  suitable  for  all  kinds 
of  bacteria.  Thus  we  are  quite  unable  to  cul- 
tivate some  species  artificially.  Others  will  only 
grow  upon  blood-serum.  Many  grow  upon  nutri- 
ent gelatine  ;  but  some  species  only  if  it  be  acid 
or  alkaline  respectively.  Whether  in  the  latter 
case  this  is  due  purely  to  tti£  reaction  or  to  the 
presence  of  the  particular  ingredients  is  an  un- 
settled point.  Though  the  comma  bacillus  of  Koch, 
like  the  majority  of  organisms,  grows  best  on  an 
alkaline  medium,  yet  the  surface  of  a  potato  is 
acid,  and  on  this  it  is  well  known  to  flourish  at 
the  temperature  of  the  blood. 

Effect  of  temperature.- — In  their  behaviour  to- 
wards temperature  bacteria  vary  considerably,  but 
still  for  the  majority  we  may  distinguish  a  maximum, 
optimum,  and  minimum  temperature. 

Many  grow  best  at  the  temperature  of  the  blood, 
and  hence  the  value  of  nutrient  agar-agar,  which 
is  not  liquefied  at  37°  C.  The  tubercle  bacillus 
will  only  grow  at  a  temperature  varying  between 
30°  and  4i°C.  On  the  other  hand,  many  forms 
grow  between  the  limits  of  5°  and  45°  C.  At 
these  temperatures  their  functional  activity  is 
paralysed,  but  they  are  not  destroyed,  for  by  re- 
moval to  favourable  conditions  they  spring  again 
to  life.  Bacteria  seem  to  have  a  special  power 


140  BACTERIOLOGY. 

of  resisting  the  effects  of  cold.  It  has  been 
stated  that  comma  bacilli  exposed  to  a  tempera- 
ture of — 10°  for  an  hour,  and  bacilli  of  anthrax 
after  exposure  to  a  temperature  of  —  no°C.,  still 
retained  their  vitality.  Temperatures  over  50°  to 
60°  C.  destroy  most  bacteria,  but  not  their  spores; 
spores  of  anthrax  retain  their  vitality  after  im- 
mersion in  boiling  water,  but  are  destroyed  by 
prolonged  boiling.  Roughly  speaking,  all  patho- 
genic bacteria  grow  best  at  the  temperature  of 
the  blood,  and  non-pathogenic  bacteria  at  the 
ordinary  temperature  of  the  room. 

Effect  of  movement.  —  Bacteria  probably  grow 
best  when  left  undisturbed.  Violent  agitation  of 
a  vessel  in  which  they  are  growing  certainly 
retards  their  growth,  but  a  steady  movement  is 
stated  not  to  affect  it ;  at  any  rate  anthrax  bacilli 
grow  with  enormous  rapidity  in  the  blood  vessels,  in 
spite  of  the  circulation. 

Effect  of  compressed  air. — Paul  Bert  maintained 
that  a  pressure  of  twenty-three  to  twenty-four 
atmospheres  stopped  all  development  of  putre- 
factive bacteria.  Oxygen,  under  a  pressure  of  five 
or  six  atmospheres,  is  stated  to  stop  their  growth. 
Other  observers  have,  however,  obtained  different 
results. 

Effect  of  gases. — Hydrogen  and  carbonic  acid  are 
stated  to  stop  the  movements  of  the  motile  bacteria. 
Chloroform  is  believed  to  arrest  the  changes  brought 
about  by  the  zymogenic  species. 


GENERAL    MORPHOLOGY   AND    PHYSIOLOGY.       14! 

Electricity. — Cohn  and  Mendelsohn  found  that 
a  constant  galvanic  current  produced  a  deleterious 
effect  owing  to  electrolysis.  At  the  positive  pole 
the  liquid  became  distinctly  acid,  and  at  the 
negative  pole  distinctly  alkaline.  With  a  weak 
current  there  appeared  to  be  no  effect,  two  power- 
ful cells  at  the  very  least  being  necessary. 

Light. — Downes  has  shown  that  sunlight  is  fatal 
to  putrefactive  bacteria.  This  is  believed  to  be 
due  to  a  process  of  induced  hyper-oxidation,  from 
which  living  organisms  ordinarily  are  shielded  by 
protective  developments  of  the  cell- wall,  or  of 
colouring  matter,  which  cut  off  injurious  rays. 
Duclaux  has  investigated  the  same  subject,  and 
observed  that  micrococci  were  more  sensitive  to 
sunlight  than  the  spore-bearing  bacilli.  Engel- 
mann  has  described  a  bacterium  whose  movements 
cease  in  the  dark,  and  Zopf  states  that  in  his 
cultures  of  Beggiatoa  roseo-persicina  the  growth  was 
much  more  strongly  developed  on  the  side  of  the 
vessel  facing  the  light. 

Chemical  reagents^ — Many  substances,  such  as 
carbolic  acid,  corrosive  sublimate,  chlorine,  bromine, 
etc.,  have  a  marked  effect  upon  the  growth  of 
bacteria.  This  will  be  more  fully  described  in 
another  chapter.  In  several  cases  the  bacteria 
themselves  secrete  a  substance  which  is  injurious 
to  their  future  development. 

Products  of  growth.  —  Bacteria  may  be 
grouped  together  according  to  the  changes  pro- 


142  BACTERIOLOGY. 

duced  in  the  media  in  which  they  grow.  Thus  we 
have  pigment-forming,  fermentative,  putrefactive, 
and  pathogenic  bacteria. 

Chromogenic  or  pigment-forming  bacteria  elabo- 
rate during  their  growth  definite  colour  stuffs. 
Such  species  are  exemplified  by  Bacillus  ianthinus, 
which  produces  a  striking  purple  growth  ; 
Bacillus  pyocyanem,  which  secretes  pyocyanin,  a 
substance  which  has  been  isolated  and  obtained 
in  a  crystalline  form  ;  Bacterium  prodigiosum,  which 
produces  a  pigment  allied  to  fuchsine ;  Beggiatoa 
roseo-persicina,  which  is  characterised  by  the 
presence  of  bacterio  -  purpurin ;  Sarcina  lutea. 
Bacillus  cyanogenus,  and  many  others. 

Zymogenic  or  ferment  bacteria  produce  their 
changes  in  non-nitrogenised  media.  Bacterium 
aceti,  by  its  growth,  produces  the  acetic  fermenta- 
tion in  wine,  by  which  alcohol  taking  up  atmo- 
spheric oxygen  is  converted  into  vinegar — 

C2H6O  +  O2  =  C2H4O2  +  H2O. 

The  fermentation  of  urine,  by  which  urea  is  con- 
verted into  carbonate,  of  ammonia,  can  be  brought 
about  by  several  micro-organisms,  but  notably 
by  the  Bacterium  urea.  The  change  produced  is 
represented  by  the  following  formula : — 

CO{NH*  +  2H2°  =  (NH4)2x:o3. 

Clostridium  butyricum  converts  the  salts  of  lactic 
acid  into  butyric  acid,  producing  the  butyric  fer- 


GENERAL    MORPHOLOGY   AND    PHYSIOLOGY.      143 

mentation  in  solutions  of  starch,  dextrine,  and 
sugar.  These  bacteria  are  agents  in  the  ripening 
of  cheese,  and  the  production  of  sauerkraut.  Thus, 
in  a  solution  neutralised  with  calcium  carbonate  :  — 


2[Ca(C3H603)2]4-H20=Ca(C4H702)2  +  CaC03  +  3CO2  +H8. 

In  the  so-called  viscous  fermentation  the  Strep- 
tococcus viscosus  produces  a  gummy  substance  in 
wines.  According  to  Pasteur,  the  change  may 
be  thus  represented  :  — 

25(C12H22On)  +  25(H2O)  =  i2(C12H20O10)  +  24(C6H14O6)  + 
i2(C02)  +  i2(H20). 

and  as  another  example  may  be  mentioned  the 
Bacillus  acidi  lactici,  through  whose  agency  sugar 
of  milk  is  converted  into  lactic  acid:  — 


Saprogenic  or  putrefactive  bacteria  play  a  most 
important  role  in  the  economy  of  nature.  They 
produce  changes  allied  to  fermentation  in  complex 
organic  substances.  The  nitrification  of  soil  has 
been  attributed  to  their  agency.  Their  action  on 
proteids,  according  to  Hoppe-Seyler,  may  be  com- 
pared to  digestion  ;  bodies  like  peptones  are  first 
produced,  then  leucin,  tyrosin,  and  fatty  acids  ; 
lastly  indol,  phenol,  sulphuretted  hydrogen,  am- 
monia, carbonic  acid,  and  water.  They  abstract 
the  elements  they  require,  and  the  remainder  enter 
into  new  combinations.  Associated  with  the  forma- 
tion of  these  substances  are  certain  bodies,  which 
have  a  poisonous  effect  when  introduced  into 


144  BACTERIOLOGY. 

animals.  These  poisonous  alkaloids,  ptomaines, 
produce  a  septic  poisoning,  which  must  be  dis- 
tinguished from  septic  infection.  The  effects  of 
septic  poisoning  depend  on  the  dose,  whereas  the 
effects  of  septic  infection  are,  to  a  certain  extent, 
independent  of  the  dose.  A  small  quantity  of  a 
septic  poison  may  produce  only  transient  effects, 
and  a  relatively  large  quantity  may  be  necessary 
to  produce  vomiting,  rigors,  and  death.  Septic 
infection,  on  the  other  hand,  may  result  equally 
from  a  small  dose,  because  the  poison  introduced 
is  a  living  organism  which  is  capable  of  propaga- 
tion and  multiplication.  Our  knowledge  of  these 
alkaloids  is  greatly  attributable  to  the  researches  of 
Selmi,  Gautier,  and  also  Brieger  and  others.  Brieger 
has  isolated  ptomaines  from  the  human  cadaver, 
putrid  meat,  fish,  and  cheese.  These  substances — 
cadaverin,  putrescin,  saprin,  peptotoxin,  and  many 
others — vary  in  their  toxic  properties. 

Pathogenic  bacteria  are  those  which  are  genetically 
related  to  disease.  Many  organisms  have  been 
supposed  to  be  pathogenic,  or  have  been  described 
in  connection  with  diseases,  which  are  only  sapro- 
phytic  associates.  By  the  latter  we  mean  organisms 
which  feed  upon  dead  organic  matter.  Such  are 
many  forms  which  are  found  on  the  skin,  in  the 
intestinal  canal,  and,  according  to  Klein,  in  the 
liver  and  internal  organs,  where  the  tissues  have 
lost  their  vitality,  and  the  organisms,  through  some 
lesion,  have  been  carried  into  the  circulation. 


GENERAL    MORPHOLOGY    AND    PHYSIOLOGY.       145 

That  many  organisms  are  causally  related  to 
disease,  there  is  strong  evidence  in  proof;  for  no 
organism  can  be  considered  to  be  productive  of 
disease  unless  it  fulfils  the  conditions  which  have 
been  laid  down  by  Koch  (p.  2).  Great  stress  must 
be  laid  upon  the  importance  of  successive  cultiva- 
tion through  many  generations,  as  the  objection 
that  a  chemical  virus  may  be  carried  over  from  the 
original  source  is  thus  overcome.  Any  hypo- 
thetical chemical  poison  carried  over  from  one 
tube  to  another,  would,  after  a  great  number  of 
such  cultivations,  be  diluted  to  such  an  immense 
extent  as  to  be  inappreciable  and  absolutely 
inert. 

Though  we  may  accept  as  a  fact  the  existence 
of  pathogenic  organisms,  we  are  not  yet  in  a  posi- 
tion to  assert  the  means  by  which  they  produce 
their  deleterious  or  fatal  effects.  Many  theories 
have  been  propounded.  It  has  been  suggested 
that  the  organisms,  micrococci  for  example,  may  be 
compared  to  an  invading  army.  The  tissue  cells 
arrayed  against  them  endeavour  to  assimilate 
and  destroy  them,  but  perish  themselves  in  the 
attempt.  This  might  explain  the  breaking  down  of 
tissue,  and  the  formation  of  local  lesions,  but  does 
not  assist  us  in  understanding  the  fatal  result  in 
thirty-six  to  forty -eight  hours  produced  by  the 
inoculation  of  the  bacilli  of  anthrax.  Another  view 
is  that  the  invading  army  seize  upon  the  commis- 
sariat, appropriating  the  general  pabulum,  which 

10 


146  BACTERIOLOGY. 

is  so  essential  to  the  life  of  the  tissues.  But  this 
would  hardly  account  for  so  acute  and  fatal  a  result 
as  anthrax,  but  would  lead  one  to  expect  symptoms 
of  inanition  and  gradual  exhaustion.  Moreover 
against  this  theory  we  have  the  fact  that  death 
may  result,  for  example,  from  anthrax,  with  the 
occasional  presence  of  comparatively  few  bacilli; 
and  again,  the  blood  may  teem  with  parasites 
such  as  the  flagellated  monads  in  well-nourished, 
healthy-looking  rats,  without  apparently  causing 
any  symptoms  whatever.  In  the  same  category 
may  be  placed  the  theory  that  eminently  aerobic 
organisms  seize  upon  the  oxygen  of  the  blood 
and  produce  death  by  asphyxia.  Another  explana- 
tion is  afforded  by  the  suggestion  of  interference 
with  the  functions  of  the  lung  and  kidney  by  mecha- 
nical blocking  of  the  capillaries.  Here  the  same 
objection  is  met  with  in  the  case  of  anthrax, 
the  same  fatal  result  may  occur  with  only  a  few 
bacilli,  while  other  cases  yield  very  beautiful 
sections,  looking  like  injected  preparations  from 
the  mapping  out  of  the  capillaries  with  the  count- 
less crowds  of  bacilli  (Plates  XVI.  and  XVII.) 

The  most  satisfactory  explanation  is  probably 
afforded  by  analogy  with  the  putrefactive  bacteria. 
We  have  seen  that  they  derive  their  necessary 
elements  from  complex  organic  substances,  and 
accompanying  the  residue  we  find  the  presence  of 
poisonous  alkaloids.  Do  pathogenic  bacteria  act 
in  the  same  way?  Does  the  anthrax  bacillus 


GENERAL    MORPHOLO3Y   AND    PHYSIOLOGY.       147 

produce  a  ptomaine  anthracin,  which  in  a  certain 
dose  produces  death,  independent  of  the  number 
of  bacilli,  provided  there  are  sufficient  present  to 
develop  that  dose?  Though  this  is  possible, 
observers  as  yet  have  failed  to  extract  from  culti- 
vations in  quantity  of  the  anthrax  bacillus  any 
alkaloid  with  virulent  properties. 

Lastly  it  has  been  suggested  that  possibly  a 
special  ferment  is  secreted  by  the  organisms,  and 
that  by  the  changes  ultimately  wrought  by  the 
action  of  this  ferment,  the  symptoms  and  phe- 
nomena of  disease  arise.  We  have  an  analogy 
with  this  theory  in  the  alkaline  fermentation  of  urine 
by  means  of  the  Toritla  urece.  By  the  researches  of 
Musculus,  and  later  of  Sheridan  Lea,  it  has  been 
shown '-that  a  ferment  is  secreted  by  the  cells  which 
can  be  isolated  in  aqueous  solution  and  is  capable 
of  rapidly  inducing  an  active  fermentation  of  urea. 
Either  of  the  two  last  theories  assists  us  in  under- 
standing how  it  is  that  in  anthrax  or  in  tuberculosis 
we  may  find  the  presence  of  only  a  few  bacilli,  or 
that,  assuming  both  tetanus  and  hydrophobia  to  be 
due  to  microbes,  we  can  have  such  a  violent  dis- 
turbance of  the  system  produced  by  the  presence 
of  very  few  micro-organisms.  We  may  conceive 
that  different  species  of  bacilli  may  vary  greatly  in 
their  power  of  producing  an  alkaloid  or  secreting 
a  ferment,  just  as  the  elaboration  of  pigment  is 
much  more  marked  in  some  species  than  in 
others ;  thus  it  need  not  follow  that  the  number  of 


148  BACTERIOLOGY. 

micro-organisms  bears  any  relation  to  the  viru- 
lence or  activity  of  the  substance  they  produce. 
There  is,  however,  yet  another  factor  in  the  pro- 
duction of  disease.  We  know  that  in  health  we 
are  proof  against  most  of  these  micro-organisms ; 
if  it  were  not  so,  we  should  all  rapidly  fall  victims 
to  the  tubercle  bacillus  or  some  others,  which  we 
in  health  inhale  with  impunity.  We  know  that  a 
microbe  may  only  cause  a  local  lesion  in  one 
animal,  and  death  in  another.  It  is  still  more 
striking  that  the  same  micro-organism,  as  is  the 
case  with  anthrax,  may  have  no  effect  whatever 
upon  certain  species  of  animals,  though  it  is 
deadly  to  others.  Again,  an  animal  naturally  sus- 
ceptible to  the  effect  of  a  pathogenic  organism 
may  be  rendered  proof  against  it.  These  matters 
will  be  discussed  in  a  future  chapter. 

Distribution  of  Bacteria. — Bacteria  are  com- 
monly described  as  ubiquitous.  They  are  ever 
present  in  the  air,  though  not  in  such  exaggerated, 
numbers  as  is  commonly  supposed.  In  nutrient 
media  exposed  to  the  air  one  is  often  astonished 
at  times  at  the  comparatively  few  bacteria  which 
develop  in  comparison  to  the  amount  of  floating 
matter,  such  as  mineral  particles,  scales,  spores  of 
fungi,  and  debris  known  to  be  present.  In  water 
they  are  also  present  in  considerable  numbers, 
though  of  course  varying  according  to  the  character 
of  the  water.  Wherever  there  is  putrefaction, 
they  are  present  in  vast  numbers.  In  the  soil,  in 


GENERAL    MORPHOLOGY    AND    PHYSIOLOGY.       149 

sewage,  in  the  intestines  ;  and  in  uncleanly  persons 
especially,  on  the  skin  and  between  the  teeth, 
various  species  may  always  be  found,  but  in  the 
healthy  blood  and  healthy  tissues  bacteria  are 
never  present.  In  a  previous  chapter  the  method 
of  examining  the  blood  of  living  persons  has  been 
described,  and  there  is,  by  this  means,  ample  oppor- 
tunity for  satisfying  oneself  that  bacteria  are  never 
to  be  found  in  the  blood  in  health.  The  organs 
removed  from  a  perfectly  healthy  animal,  with  the 
necessary  precautions,  into  sterilised  media  can  be 
kept  indefinitely  without  undergoing  putrefaction, 
or  giving  any  development  of  bacteria.  This  has 
been  established  by  many  observers,  notably 
Cheyne  and  Hauser;  and  the  results  of  former 
observers  to  the  contrary  must  be  attributed  to 
imperfect  methods  admitting  of  accidental  con- 
tamination. 


CHAPTER  IX. 

ANTISEPTICS    AND    DISINFECTANTS. 

IN  the  previous  chapter  several  conditions  were 
alluded  to  which  affected  the  growth  of  bacteria, 
such  as  the  nature  of  the  nutrient  soil,  temperature, 
light,  and  electricity.  The  effect  of  certain  chemi- 
cal substances,  and  of  excessive  heat  and  cold, 
was  also  mentioned,  but  this  constitutes  a  subject 
of  such  practical  importance  that  it  must  be  con- 
sidered more  fully. 

Agents  which  retard  the  growth  of  bacteria  are 
generally  spoken  of  as  antiseptics  as  distinguished 
from  disinfectants,  which  altogether  destroy  their 
vitality. 

Though  chemical  disinfectants,  or  germicides, 
when  diluted,  act  as  efficient  antiseptics,  the  con- 
verse, that  an  antiseptic  in  a  sufficiently  concentrated 
form  will  act  as  a  disinfectant,  is  not  the  case.  The 
term  "antiseptic,"  indeed,  should  be  restricted  to 
those  substances  or  agents  which  arrest  the  changes 
bacteria  produce,  but  which  do  not  prevent  their 
springing  into  activity  when  removed  to  favourable 
conditions.  Thus  excessive  heat,  which  destroys 


ANTISEPTICS   AND    DISINFECTANTS.  15! 

bacteria  and  their  spores,  is  a  true  disinfectant ;  and 
excessive  cold,  which  only  benumbs  them,  retard- 
ing- their  development  without  killing  them,  is  an 
antiseptic. 

Spores  have  a  greater  power  of  resisting  the  action 
of  these  various  agents  than  the  parent  cells,  and 
many  species  of  micro-organisms  differ  from  each 
other  in  their  resisting  power.  An  exact  knowledge 
of  the  subject  can,  therefore,  only  be  based  upon 
investigations  which  will  determine  the  effect  of 
these  agents  upon  pure  cultivations  of  the  different 
micro-organisms  causally  related  to  putrefaction 
and  disease.  In  the  latter  case,  especially,  this  is 
not  possible  in  the  present  state  of  our  knowledge. 
In  some  cases  of  communicable  disease  there  is 
considerable  doubt  as  to  the  etiological  importance 
of  the  organisms  which  have  been  described ;  in 
other  cases  no  organisms  have  as  yet  been  dis- 
covered, or  the  organisms  -cannot  be  artificially 
cultivated,  or  the  disease  is  not  reproduced  by 
inoculation,  so  that  there  is  no  means  of  testing 
whether  the  agents  have  had  any  effect.  One  can, 
therefore,  only  draw  general  conclusions  by  selecting 
some  well-known  pathogenic  and  non-pathogenic 
micro-organisms,  and  considering  the  influence  of 
chemicals,  of  hot  air,  and  of  steam  upon  them,  as 
representing  the  effect  upon  the  various  contagia 
of  disease  and  the  causes  of  putrefaction. 

Such  knowledge  must  necessarily  prove  of  the 
greatest  importance, — to  the  sanitarian,  who  is  con- 


1 52  BACTERIOLOGY. 

cerned  in  preventing  the  spreading  of  disease 
and  in  the  disposal  of  putrefactive  matter, — to  the 
surgeon,  who  is  anxious  to  exclude  micro-organisms 
during  surgical  operations,  and  to  arrest  the 
development  in  wounds  of  bacteria  which  have 
already  gained  an  entrance, — to  the  physician  in 
the  treatment  of  micro-parasitic  diseases.  The 
sanitarian  and  the  surgeon  must  profit  directly 
by  such  experiments,  for  in  the  disinfection  of 
clothes  and  the  sick-room  by  the  one,  and  in 
the  application  of  antiseptic  dressings  and  lotions 
by  the  other,  the  micro-organisms  are  encoun- 
tered as  in  the  test  experiments  apart  from  the 
living  body.  The  physician,  on  the  other  hand,  is 
principally  concerned  in  dealing  with  micro-para- 
sites when  circulating  in  the  blood,  or  carrying  on 
their  destructive  processes  in  the  internal  tissues. 
So  far  as  our  knowledge  at  present  goes,  the 
physician  can  avail  himself  but  little  of  the  effect 
of  the  direct  application  of  the  substances  which 
have  been  found  to  retard  or  destroy  the  growth 
of  the  organisms  in  artificial  cultivations,  for  the 
concentrated  form  in  which  they  would  have  to  be 
administered  would  prove  as  deleterious  or  as  fatal 
to  the  host  as  to  the  parasites.  Thus  Koch  has 
stated  that  to  check  the  growth  of  the  anthrax 
bacillus  in  man  it  would  be  necessary  that  there 
should  be  twelve  grammes  of  iodine  constantly  in 
circulation ;  and  that  the  dose  of  quinine  necessary 
to  destroy  the  spirilla  of  relapsing  fever  would  be 


ANTISEPTICS    AND    DISINFECTANTS.  153 

from  twelve  to  sixteen  grammes.  The  retarding 
influence,  however,  of  certain  substances  when 
diluted,  and  the  fact  that  disinfectants  are  some- 
times equally  efficacious  in  a  diluted  form  when 
their  application  is  prolonged,  seem  to  indicate 
measures  which  may  be  adopted,  in  some  cases, 
with  chances  of  success,  such  as  the  inhalation  of 
antiseptic  vapours  in  phthisis.  For  the  most  part 
the  physician  must  look  rather  to  combating  the 
effects  of  micro-organisms  by  restoring  to  its 
normal  standard  the  lowered  vitality  which  enabled 
the  bacteria  to  get  a  footing. 

There  is  no  wider  field  for  research  than  the 
determination  of  the  real  effect  of  disinfectants 
and  antiseptics.  Painstaking  and  laborious  as 
the  researches  are  which  have  been  hitherto 
made,  the  subject  is  so  beset  with  fallacies, 
leading,  in  some  cases,  to  totally  erroneous  conclu- 
sions, that  it  is  not  surprising  that  one  meets  on 
all  sides  with  conflicting  statements.  The  author 
has  no  intention  of  analysing  these  results,  but  a 
general  idea  will  be  given  of  the  methods  which 
have  been  employed,  and  for  further  details 
reference  must  be  made  to  the  original  papers 
mentioned  in  the  bibliography. 

Chemical  substances. — It  was  customary  to  judge 
of  the  power  of  a  disinfectant  or  antiseptic  by 
adding  it  to  some  putrescent  liquid.  A  small  por- 
tion of  the  latter  was,  after  a  time,  transferred  to 
some  suitable  nourishing  medium,  and  the  efficacy 


154  BACTERIOLOGY. 

of  the  substance  estimated  by  the  absence  of 
cloudiness,  odour,  or  other  sign  of  development 
of  bacteria  in  the  inoculated  fluid.  Koch  pointed 
out  the  errors  that  might  arise  in  these  experiments 
from  accidental  contamination,  or  from  there  being 
no  evidence  of  the  destruction  of  spores ;  and  we 
are  indebted  to  him  for  a  complete  and  careful 
series  of  observations  with  more  exact  methods. 

Instead  of  employing  a  mixture  of  bacteria, 
Koch's  plan  was  to  subject  a  pure  cultivation  of 
some  well-known  species  with  marked  character- 
istics to  the  reagent  to  be  tested.  A  small 
quantity  was  then  transferred  to  fresh,  nourishing 
soil,  under  favourable  conditions,  side  by  side  with 
nutrient  material  inoculated  from  a  cultivation 
without  treatment  with  the  disinfectant.  The  latter 
constituted  a  control  test,  which  is  most  essential  in 
all  such  experiments.  To  test  the  resistant  power 
of  bacteria  which  are  easily  destroyed,  two  species 
were  selected,  the  so-called  Micrococcus  prodigiosus, 
and  the  bacillus  of  blue  pus.  These  were  culti- 
vated on  potatoes,  the  surface  of  which  was  sliced 
off  and  dried.  A  fragment  transferred  to  freshly- 
prepared  potato  gave  rise  to  a  growth  of  the 
particular  micro-organism ;  but  if  after  treatment 
with  some  reagent  no  growth  occurred,  the  conclu- 
sion was  drawn  that  the  agent  was  efficacious  in 
destroying  the  vitality  of  the  bacteria. 

Anthrax  bacilli  in  blood  withdrawn  from  an 
animal  just  killed  were  taken  to  represent  spore- 


ANTISEPTICS    AND    DISINFECTANTS,  155 

less  bacteria,  while  silk  threads  steeped  in  an 
artificial  cultivation  of  the  bacilli  and  dried,  afforded 
a  means  of  testing  the  vitality  of  spores. 

Even  by  employing  pure  cultivations  on  solid 
media,  great  precautions  were  necessary  to  avoid 
mistakes.  If,  for  instance,  a  large  quantity  of  the 
growth  which  had  been  subjected  to  some  chemical 
solution  were  carried  over  to  the  fresh  tube  con- 
taining the  nutrient  medium,  or  if  a  silk  thread, 
which  had  been  dipped  in  a  solution,  were  directly 
transferred  to  the  new  soil,  enough  of  the  supposed 
disinfectant  might  be  mechanically  carried  over  to 
retard  the  development  of  the  bacteria,  though 
it  was  ineffectual  in  destroying  them.  From  a 
growth  not  appearing,  the  conclusion  might  be 
drawn  that  the  spores  or  the  bacteria  had  been 
affected,  and  so  a  mistake  occurs.  To  avoid  this 
Koch  made  a  point  of  transferring  a  minimum  of 
the  disinfected  growth  to  as  large  a  cultivation 
area  as  possible,  so  that  any  chemical  substance 
mechanically  carried  over,  would  be  so  diluted  as 
to  be  inert.  For  the  same  reason  threads,  after 
withdrawal  from  the  disinfecting  solution,  were 
rinsed  in  sterilised  water,  or  weak  alcohol,  and 
then  transplanted ;  or,  instead  of  judging  from  the 
development  on  nutrient  gelatine,  the  effect  of 
inoculation  in  a  healthy  animal  was  made  the  test. 

A  few  examples  may  be  quoted  in  illustration. 
Silk  threads,  impregnated  with  anthrax  spores, 
were  placed  in  bottles  containing  carbolic  acid  of 


156  BACTERIOLOGY. 

various  strengths.  A  thread  was  removed  from 
each  on  successive  days,  and  transferred  to  nutrient 
gelatine,  and  the  result  noted.  It  was  found  that 
immersion  of  the  thread  in  a  5  per  cent,  solution 
of  carbolic  acid  was  sufficient  in  two  days  to  effect 
complete  sterilisation,  and  seven  days  in  a  3  per 
cent,  solution  was  equally  efficacious.  Since  for 
practical  purposes  a  strength  should  be  selected 
which  would  be  effectual  in  twenty-four  hours, 
Koch  recommended  that  for  general  use,  allow- 
ing for  deterioration  by  keeping,  a  solution  con- 
taining not  less  than  5  per  cent,  should  be 
employed,  and  for  complex  fluids  probably  a  still 
higher  percentage  would  be  necessary.  In  the 
case  of  sporeless  bacilli  the  results  were  very 
different.  Blood,  containing  the  bacilli,  from  an 
animal  just  killed,  was  dried  on  threads,  and  after 
exposure  for  two  minutes  to  a  i  per  cent,  solution, 
was  completely  sterilised.  Fresh  blood  mixed  with 
a  i  per  cent,  carbolic  solution  produced  no  effect  on 
inoculation.  If,  on  the  other  hand,  the  blood  was 
mixed  with  a  "5  per  cent,  solution,  the  virulence 
was  not  destroyed.  The  facility  with  which  the 
bacilli  are  destroyed,  compared  with  their  spores, 
illustrates  how  easily  errors  may  occur,  if  mere 
arrest  of  growth  or  loss  of  motility  be  regarded  as 
a  sign  of  the  efficacy  of  disinfection. 

To  test  vapours,  Koch  exposed  anthrax  spores 
or  the  spores  which  occur  in  garden  earth  by  sus- 
pending them  over  solutions,  e.g.,  of  bromine  or 


ANTISEPTICS    AND    DISINFECTANTS.  157 

chlorine  in  a  closed  vessel.  After  a  time  they  were 
transferred  to  a  nutrient  medium  to  test  their  vitality. 
To  test  the  power  of  sulphurous  acid  gas,  the  spores 
were  spread  about  in  a  room  in  which  the  gas  was 
generated  by  burning  sulphur  in  the  ordinary  way 
for  disinfecting  a  room.  To  test  chemicals  which 
might  be  recommended  for  disinfecting  vans  and 
railway  carriages,  spores  were  laid  on  boards  which 
were  then  washed  or  sprayed,  and  the  spores  then 
transferred  to  the  nutrient  gelatine. 

By  such  simple  methods  Koch  investigated  a 
long  list  of  chemical  reagents,  and  according  to 
these  experiments  the  salts  of  mercury,  and  the 
chloride  especially,  proved  most  valuable.  Where 
heat  is  not  admissible,  these  compounds  were 
therefore  highly  recommended,  though  their  poi- 
sonous nature  is  a  drawback  to  their  indiscrimi- 
nate use.  Koch  states,  for  disinfecting  a  ship's 
bilge,  where  a  5  per  cent,  solution  of  carbolic 
acid  must  be  left  for  forty-eight  hours,  a  i  in  1000 
solution  of  mercuric  chloride  would  only  require  a 
few  minutes. 

There  is,  on  the  other  hand,  reason  for  doubting 
the  efficacy  of  mercuric  chloride ;  for,  though 
anthrax  spores  subjected  to  a  i  in  20,000  solution 
of  mercuric  chloride  for  ten  minutes,  and  then 
washed  in  alcohol,  gave  no  growth  in  nutrient 
gelatine,  silk  threads  exposed  for  ten  minutes  to 
a  i  in  20,000  solution,  or  even  i  in  10,000,  still 
proved  fatal  to  mice. 


158  BACTERIOLOGY. 

Herroun  considers  that  the  value  of  mercuric 
chloride  as  an  antiseptic  is  much  over-rated,  as 
he  has  cultivated  ordinary  septic  bacteria  in 
albuminous  filtrates,  containing  i  in  2,000.  It  is 
precipitated  by  albumins  if  used  of  greater  strength, 
and  is  readily  converted  by  the  sulphur  of  albu- 
minous bodies  into  mercuric  sulphide, — a  com- 
pound which  has  practically  no  antiseptic 
properties. 

Sternberg  has  also  made  an  elaborate  series  of 
experiments  with  regard  to  the  action  of  germi- 
cides. In  this  case  cultivations  of  well-known 
pathogenic  organisms  in  liquid  media  were  em- 
ployed. The  supposed  germicide  was  added  to  the 
liquid  cultivation,  and  after  two  hours  a  fresh  flask 
of  sterilised  culture  was  inoculated  from  the  dis- 
infected cultivation,  and  placed  in  the  incubator. 
In  twenty- four  to  forty-eight  hours,  if  the  chemical 
was  not  efficient,  there  was  evidence  of  a  growth  ot 
bacteria.  Blyth  has  investigated  the  disinfection 
of  cultivations  of  Bacterium  termo,  of  sewage,  and 
typhoid  excreta,  and,  in  conjunction  with  Klein,  the 
effect  of  well-known  disinfectant  materials  on  an- 
thrax spores.  Miquel,  Laws,  and  others,  have  also 
contributed  to  our  knowledge  of  the  effect  of  anti- 
septics and  disinfectants  upon  micro-organisms. 
In  spite  of  all  that  has  been  done,  there  is  room  for 
many  workers ;  a  great  deal  of  ground  must  be 
gone  over  again  to  rectify  discrepancies,  examine 
conflicting  results,  and  thus  determine  what 


ANTISEPTICS    AND    DISINFECTANTS.  159 

observations  may  be  relied  upon  for  practical 
application. 

Hot  Air  and  Steam. — Koch,  in  conjunction  with 
Wolfhugel,  also  tested  the  value  of  hot  air.  A 
similar  plan  was  adopted  as  in  disinfection  with 
chemicals.  Bacteria  and  spores  were  subjected  for 
a  certain  time  to  a  known  temperature  in  a  hot- 
air  chamber,  and  then  were  transferred  to  a 
nourishing*  soil,  or  animals  were  inoculated. 

Paper  parcels,  blankets,  bags,  and  pillows,  con- 
taining samples  of  micro-organisms  wrapped  up 
inside,  were  also  placed  in  the  hot-air  chamber,  to 
test  the  power  of  penetration  of  heat. 

The  conclusions  from  such  experiments  were 
as  follows: — 

Sporeless  micro-organisms  at  a  little  over  100°  C. 
are  destroyed  in  one  hour  and  a  half. 

Spores  of  bacilli  require  three  hours  at  140°  C. 

If  enclosed  in  pillows  and  blankets,  exposure 
from  three  to  four  hours  to  140°  C.  is  necessary. 

Spores  of  fungi  require  one  and  a  half  hours  at 
1 10 — 115°  C. 

Further  experiments  showed  that  at  the  tempera- 
ture necessary  for  the  destruction  of  spores  of 
bacilli  almost  all  fabrics  are  more  or  less  injured. 

Koch,  in  conjunction  with  Gaffky  and  Loffler, 
also  investigated  the  effect  of  steam  under  pressure 
and  at  the  atmospheric  pressure. 

Rolls  of  flannel  with  anthrax  spores  or  earth 
spores,  and  a  thermometer  wrapped  up  inside,  were 


160  BACTERIOLOGY. 

subjected  to  steam,  and  the  results  compared  with 
the  effect  obtained  with  hot  air. 

Thus  in  hot  air  four  hours'  exposure  to  a 
temperature  of  130°  C. — 140°  C.  brought  the 
temperature  inside  the  roll  to  85°  C.,  and  the 
spores  were  not  injured  ;  on  the  other  hand,  ex- 
posure to  steam  under  pressure  at  i2o°C.  for  one 
and  a  half  hours,  raised  the  internal  temperature 
to  117°  C.  and  killed  the  spores. 

By  such  experiments  the  superior  penetrative 
power  of  steam-heat  was  established. 

To  test  steam-heat  at  the  atmospheric  pressure, 
water  was  boiled  in  a  glass  flask  with  its  neck 
prolonged  by  means  of  a  glass  tube,  the  tempera- 
ture in  which  was  found  to  be  uniform  throughout. 
Anthrax  and  earth  spores  placed  in  the  tube  were 
found  to  be  unable  to  withstand  steam  at  100°  C. 
even  for  a  few  minutes.  It  was,  therefore,  concluded 
that  disinfection  by  steam  at  atmospheric  pressure 
was  superior  to  hot  air  from  its  greater  efficiency, 
and  to  steam  under  pressure  from  the  simplicity 
of  the  necessary  apparatus. 

Parsons  and  Klein  made  some  experiments  which 
were  more  in  favour  of  dry  heat  than  the  above. 
These  observers  state  that  anthrax  bacilli  are 
destroyed  by  an  exposure  of  five  minutes  to  from 
100°  C.  to  103°  C.,  and  that  anthrax  spores  are  de- 
stroyed in  four  hours  at  104°  C.,  or  in  one  hour  at 
1 1 8°  C.  Guinea-pigs  inoculated  with  tuberculous  pus 
which  had  been  exposed  for  five  minutes  to  104°  C., 


ANTISEPTICS   AND   DISINFECTANTS.  l6l 

remained  unaffected.  They  concluded  that  as  none 
of  the  infectious  diseases,  for  which  disinfecting 
measures  are  in  practice  commonly  applied,  are 
known  to  depend  upon  the  presence  of  bacilli  in 
a  spore-bearing  condition,  their  contagia  are  not 
likely  to  retain  their  activity  after  being  heated 
for  an  hour  to  105°  C.  (220°  F.). 

In  experiments  with  steam,  the  results  were  in 
accordance  with  those  already  given,  and  complete 
penetration  of  an  object  by  steam-heat  for  more 
than  five  minutes  was  deemed  sufficient.  They  also 
arrived  at  the  same  result  as  in  Koch's  experiments, 
that  steam -chambers  are  preferable  to  those  in 
which  dry  heat  is  employed,  though  it  must  be 
borne  in  mind  that  some  articles,  such  as  leather, 
are  injured  by  exposure  to  steam. 


ii 


CHAPTER  X. 

IMMUNITY. 

THE  condition  of  being  insusceptible  to  an 
infective  disease  may  be  either  natural  or  acquired. 
In  the  description  of  the  pathogenic  organisms 
several  examples  of  natural  immunity  will  be  en- 
countered. The  bacillus  of  septicaemia,  so  fatal 
to  house  mice,  has  been  shown  to  have  no  effect 
upon  field  mice.  The  bacillus  of  anthrax  is 
innocuous  to  pigs,  cats,  white  rats,  and  to  adult 
dogs,  asses,  and  horses.  The  bacterium  of  rabbit 
septicaemia  is  equally  inert  in  dogs,  rats,  and 
guinea-pigs.  The  immunity  may  be  as  in  these 
cases  complete,  or  only  partial.  Ordinary  sheep 
are  very  easily  affected  with  anthrax,  but  Algerian 
sheep  only  succumb  to  large  doses  of  the  virus. 
Natural  immunity  may  not  only  be  characteristic 
of  certain  species,  but  it  may  occur  in  certain 
individuals  of  a  susceptible  species.  The  same 
occurs  in  man,  for  certain  individuals,  though 
equally  exposed  during  an  epidemic  of  small-pox, 
may  escape  where  others  readily  fall  victims  to 
the  disease. 


IMMUNITY.  163 

Acquired  immunity  is  illustrated  by  the  protec- 
tion afforded  by  one  attack  of  the  exanthemata 
against  subsequent  attacks.  Thus  one  attack  of 
measles  or  small-pox,  as  a  rule,  affords  complete 
protection.  A  knowledge  of  the  immunity  result- 
ing in  the  latter  case  led  to  the  introduction  of 
inoculation  of  small-pox  prior  to  the  establishment 
by  Jenner  of  the  protective  influence  of  vaccination. 

Immunity  may  be  acquired  by  acclimatization, 
for  the  inhabitants  of  tropical  climates  are  less 
susceptible  to  the  diseases  of  the  country,  malarial 
fevers  for  instance,  than  strangers. 

In  civilised  communities  also  there  appears  to  be 
a  degree  of  acquired  immunity,  for  the  infectious 
diseases  introduced  among  savages  or  isolated 
communities  have  assumed  the  most  virulent 
properties. 

The  immunity  acquired  by  protective  inocula- 
tion constitutes,  in  connection  with  the  study  of 
pathogenic  micro-organisms,  a  subject  of  pre- 
eminent interest  and  importance.  Pasteur,  in  his 
researches  upon  fowl-cholera,  observed  that  after 
non-fatal  cases  the  disease  either  did  not  recur, 
or  the  severity  of  a  subsequent  attack  was  in 
inverse  proportion  to  the  severity  of  the  first 
attack*  It  occurred  to  him  to  endeavour  to  obtain 
the  virus  of  this  disease  in  a  form  which  would 
provoke  a  mild  attack  of  the  disease,  and  thus 
give  protection  against  the  virulent  form.  This 
attenuation  or  mitigation  of  the  virus  was 


1 64  BACTERIOLOGY. 

successfully  attained  in  the  following-  manner: 
Cultivations  of  the  microbe,  in  chicken-broth,  were 
allowed  to  remain  with  a  lapse  of  several  months 
between  the  carrying  on  of  successive  cultivations 
in  fresh  media.  The  new  generations  which  were 
then  obtained  were  found  to  have  diminished  in 
virulence,  and  ultimately  a  virus  was  obtained 
which  produced  only  a  slight  disorder;  on  re- 
covery the  animal  was  found  to  be  proof  against 
inoculation  with  virulent  matter.  The  explanation 
given  by  Pasteur  of  this  change  was,  that  prolonged 
contact  with  the  oxygen  of  the  air  was  the  influence 
which  diminished  the  virulence,  and  he  endeavoured 
to  prove  this  by  showing  that  if  broth  were  in- 
oculated in  tubes  which  could  be  sealed  up,  so 
that  only  a  small  quantity  of  air  was  accessible 
to  the  microbe,  the  virulence  of  the  cultures  was 
retained. 

Toussaint  investigated  the  possibility  of  attenuat- 
ing the  virus  of  anthrax.  Sheep  injected  with 
3  ccm.  of  defibrinated  blood,  containing  anthrax 
bacilli,  which  had  been  exposed  to  55°  C.  for  ten 
minutes,  recovered,  and  were  afterwards  insuscep- 
tible. Pasteur  subsequently  argued  that  this 
method  did  not  admit  of  practical  application ; 
difficulties  would  arise  in  dealing  with  infective 
blood  in  quantity,  and  artificial  cultivations  started 
from  this  blood  could  not  be  relied  upon,  as 
they  proved  sometimes  as  virulent  as  ever. 

Pasteur  endeavoured  to  apply  the  same  method 


IMMUNITY  165 

for  obtaining  an  attenuated  virus  of  anthrax,  as 
he  had  successfully  employed  in  chicken-cholera. 
A  difficulty  was  soon  encountered,  for  in  cultiva- 
,tions  of  the  bacillus  with  free  access  of  air  spore- 
formation  readily  takes  place,  and  the  spores  are 
well  known  to  have  an  extraordinary  power  of 
retaining  their  virulence.  Pasteur  found  that  the 
bacilli  ceased  to  develop  at  45°  C.,  and  he  believed 
that  spore-formation  ceased  at  42° — 43°  C.,  the 
bacilli  continuing  to  develop  by  fission  only.  The 
cultivations  were,  therefore,  kept  at  this  tempera- 
ture, and  at  the  end  of  eight  days  the  bacilli  were 
found  to  have  lost  their  virulence,  and  were  quite 
inert  when  inoculated  in  guinea-pigs,  sheep.,  or 
rabbits.  This  total  destruction  was,  however, 
preceded  by  a  gradual  mitigation,  so  that  a  virus 
could  be  obtained,  by  taking  it  at  the  right  time, 
which  only  gave  a  mild  disease,  and  afforded 
subsequent  protection. 

At  Melun,  in  1881,  the  protective  inoculation 
against  anthrax  was  put  to  a  practical  test.  Sheep 
and  oxen  were  inoculated  with  the  mitigated  virus, 
and  then  with  a  virulent  form ;  at  the  same  time 
other  sheep  and  oxen  were  inoculated  with  the 
virulent  form  without  previous  vaccination  as  a 
control  experiment.  The  unprotected  sheep  died 
without  exception  ;  the  unprotected  oxen  suffered 
from  cedematous  swellings  at  the  seat  of  inocu- 
lation, and  a  rise  of  temperature ;  but  all  the 
protected  animals  remained  healthy. 


1 66  BACTERIOLOGY. 

As  a  result  of  these  experiments  an  idea  arose 
that  by  preventive  inoculation  with  attenuated 
virus  all  communicable  diseases  would  in  time  be 
eradicated ;  but  this  does  not  follow,  for  all  com- 
municable diseases  do  not  confer  immunity  after 
a  first  attack,  and  in  some  cases  the  very  reverse 
is  believed  to  occur.  Thus  erysipelas  of  the 
face  leads  to  an  increased  liability  to  subsequent 
attacks  of  the  same  disease.  Again,  the  occurrence 
of  one  disease  is  stated  to  induce  a  liability  to 
others  ;  small-pox  and  typhoid  fever  are  regarded 
as  predisposing  to  tuberculosis ;  so  that  the 
principle  of  preventive  inoculation  does  not  apply 
in  these  cases,  and  its  effect  would  probably  tend 
rather  to  deleterious  results  than  otherwise.  Even 
with  regard  to  the  prevention  of  anthrax,  Pasteurs 
researches  were  opposed  and  criticised.  Koch 
investigated  the  subject,  and  came  to  the  con- 
clusion that  the  process  did  not  admit  of  practical 
application,  chiefly  on  the  ground  that  as  immunity 
only  lasted  a  year,  the  losses  from  the  vaccination 
process  would  be  as  great  or  even  greater  than 
from  the  spontaneous  disease ;  further,  there  was 
danger  in  disseminating  a  vaccine  of  the  strength 
required  to  be  effectual.  Chauveau  proved  that 
the  attenuation  was  due  to  the  temperature,  and 
not  to  the  prolonged  effect  of  oxygen.  By  keeping 
cultivations  at  42° — 43°  C.  in  vacua,  the  virulence  was 
found  to  disappear  in  twenty-four  hours,  and  by 
keeping  cultivations  at  a  low  temperature  with  free 


IMMUNITY.  167 

access  of  air  the  virulence  was  retained.  Chauveau 
considered,  there! ore,  not  only  that  oxygen  was 
not  the  agent,  but  that  the  mitigation  was  much 
more  easily  effected  in  its  absence.  In  spite  of 
these  adverse  criticisms,  these  researches  never- 
theless confirmed  the  principle  of  Pasteur's  con- 
clusion, that  immunity  could  be  induced  by 
experimental  measures,  and  further  showed  that 
he  had  considerably  advanced  the  method  by 
which  this  could  be  effected, 

Chauveau  succeeded  also  in  attenuating  the  virus 
by  a  modification  of  Toussaint's  method.  Sterilised 
broth  was  inoculated  with  the  bacilli,  and  placed 
in  the  incubator  at  42° — 43°  C.  After  the  lapse  of 
twenty  hours  it  was  removed  to  another  incubator 
at  47°  C.  According  to  the  time  of  exposure  to  this 
increased  temperature,  the  mitigation  varied  in  de- 
gree. Thus  inoculation  with  the  virus,  before  it  was 
exposed  to  47° C.,  was  fatal  to  guinea-pigs;  but 
after  one  hour  at  47°  C.  the  virulence  was  diminished, 
and,  though  ultimately  fatal,  life  was  prolonged  ; 
after  two  hours'  exposure  at  47°  C.  only  half  the 
animals  died  ;  and  after  three  hours'  exposure  they 
recovered  and  were  rendered  refractory  to  sub- 
sequent inoculation. 

Attenuation  of  the  virus  has  also  been  induced 
by  chemical  means.  Chamberland  and  Roux 
stated  that  a  fresh  growth  started  from  a  cultiva- 
tion of  bacilli  which  had  been  subjected  for  twenty- 
nine  days  to  &Jo  of  carbolic  acid  was  found  to 


1 68  BACTERIOLOGY. 

be  inert  in  guinea-pigs  and  rabbits.  Bichromate 
of  potash  added  to  a  cultivation  in  the  proportion 
of  TTUOO  —  TOGO  £ave»  after  three  days,  a  new 
growth,  which  killed  rabbits,  guinea-pigs,  and  half 
the  sheep  inoculated;  after  ten  days,  rabbits  and 
guinea-pigs,  but  not  sheep ;  and  after  a  longer 
time  even  guinea-pigs  were  unaffected. 

In  other  diseases  similar  results  have  been 
obtained. 

Arloing,  Cornevin,  and  Thomas  found  that  by 
inoculating  a  small  quantity  of  the  virus  of  symp- 
tomatic anthrax  anywhere  in  the  subcutaneous 
connective  tissue,  or  a  moderate  quantity  at  the 
root  of  the  tail,  and  even  by  intravenous  injection, 
immunity  was  obtained  from  a  virulent  dose. 

In  swine-erysipelas,  Pasteur  and  Thuillier  ob- 
tained attenuated  virus  upon  quite  another  principle. 
They  discovered  that  by  passing  the  virus  through 
pigeons  the  virulence  was  increased,  but  by  passing 
it  through  rabbits  it  was  progressively  diminished. 
Thus  a  virus  was  obtained  from  the  rabbit,  which 
produced  only  a  mild  disease  in  pigs,  and  after 
recovery  complete  immunity.  Similarly  in  rabies 
Pasteur  finds  that  passage  of  the  virus  through 
various  animals  considerably  modifies  its  properties. 
By  inoculating  a  monkey  from  a  rabid  dog,  and 
then  passing  the  virus  through  other  monkeys,  the 
virulence  is  diminished ;  but  by  inoculating  a  rabbit 
from  the  dog,  and  passing  the  virus  from  rabbit 
to  rabbit,  the  virulence  is  increased.  More  recently 


IMMUNITY.  169 

Pasteur  has  employed  another  method  of  attenuat- 
ing the  virus  of  rabies.  The  spinal  cord  of  inoculated 
rabbits  is  removed  with  all  possible  precautions,  and 
portions  a  few  centimetres  in  length  are  suspended 
in  flasks  in  which  the  air  is  dried  by  fragments 
of  potash.  By  this  process  the  virulence  is  found 
to  gradually  diminish  and  finally  disappear. 

Animals  inoculated  with  portions  of  these  cords, 
after  suspension  for  a  certain  time,  are  rendered 
refractory  to  inoculation  with  virulent  cords. 
Having  rendered  dogs,  which  had  been  previously 
bitten,  free  from  the  supervention  of  symptoms  of 
hydrophobia  by  means  of  protective  inoculation, 
Pasteur  proceeded  to  apply  the  same  treatment  to 
persons  bitten  by  rabid  animals,  with  results  which 
tend  to  the  belief  that  a  prophylactic  for  rabies  has 
been  found,  though  this  must  still  be  considered  to 
be  sub  judice. 

The  question  as  to  what  constitutes  immunity 
is  a  vexed  one. 

Raulin  has  shown  that  Aspergillus  niger  develops 
a  substance  which  is  prejudicial  to  its  own  growth 
in  the  absence  of  iron  salts  in  the  nutrient  soil. 
Pasteur  has  suggested  that  in  rabies  side  by  side 
with  the  living  and  organised  substance  there  is 
some  other  substance  which  has,  as  in  Raulin's 
experiment,  the  power  of  arresting  the  growth 
of  the  first  substance.  If  we  accept  the  theory 
of  arrest  by  some  chemical  substance,  we  must 
suppose  that  in  the  acquired  immunity  afforded  by 


I7O  BACTERIOLOGY. 

one  attack  of  an  infectious  disease  this  chemical 
substance  is  secreted,  and,  remaining  in  the  system, 
opposes  the  onset  of  the  micro-organism  at  a  future 
time.  In  the  natural  immunity  of  certain  species 
and  individuals  we  must  suppose  that  this  chemical 
substance  is  normally  present. 

Another  theory  is,  that  the  micro-organisms 
assimilate  the  elements  which  they  require  for  their 
nutrition  from  the  blood  and  tissues,  and  render 
the  soil  impoverished  or  otherwise  unsuitable  for 
the  development  of  the  same  micro-organisms  here- 
after ;  this  condition  may  be  permanent,  or  the 
chemical  constitution  of  the  tissues  may  be  restored 
to  normal,  when  immunity  ceases.  If,  however, 
we  explain  acquired  immunity  by  the  result  of  the 
growth  of  a  previous  invasion  of  micro-organisms, 
we  are  still  confronted  with  the  difficulty  of  explain- 
ing natural  immunity. 

A  third  theory  is  that  the  tissues  are  endowed 
with  some  power  of  vital  resistance  to  the  develop- 
ment of  micro-organisms,  similar  to  the  vital 
resistance  to  the  coagulation  of  the  blood,  which  is 
supposed  to  exist  in  the  lining  membrane  of  the 
healthy  blood-vessel ;  that  in  some  species  and  indi- 
viduals this  exists  to  a  high  degree,  and  hence 
their  natural  immunity  But  this  does  not  explain 
how  one  attack  confers  immunity  from  a  subsequent 
one.  One  would  expect  that  the  vital  resistance 
would  invariably  be  lowered  by  a  previous  attack, 
and  increased  liability  be  the  constant  result. 


IMMUNITY.  I  7  I 

Lastly,  that  leucocytes  appear  to  have  the  power 
of  destroying  bacteria  in  some  cases,  has  been 
demonstrated  by  the  researches  of  Metschnikoff, 

If  anthrax  bacilli  are  inoculated  in  the  frog,  the 
white  blood- cells  are  observed  to  incorporate  and 
destroy  them  until  they  entirely  disappear,  and  the 
animal  is  not  affected.  But  if  the  animal,  after 
inoculation,  is  kept  at  a  high  temperature,  the 
bacilli  increase  so  rapidly  that  they  gain  the  upper 
handover  the  leucocytes,  and  the  animal  succumbs. 

In  septicaemia  of  mice  the  white  blood-cells  are 
attacked  and  disintegrated  by  the  bacilli  in  a 
similar  way.  It  is  difficult,  however,  to  accept  any 
explanation  of  immunity  from  these  observations, 
— to  suppose,  for  example,  that  immunity  depends 
upon  the  micro-organisms  being  unable  to  cope 
with  the  leucocytes  in  certain  species.  It  is  difficult 
to  conceive  that  the  leucocytes  in  the  blood  and 
tissues  in  the  field  mouse  are  differently  constituted 
from  those  in  the  house  mouse,  so  that  they  form 
an  effectual  barrier  in  the  one  case,  though  so 
readily  destroyed  in  the  other. 


PART    III. 

SYSTEMATIC  AND    DESCRIPTIVE,   WITH    SPECIAL 
MICROSCOPICAL  METHODS. 


CHAPTER   XI. 

CLA  SSIFICA  TION  OF  BA  CTERIA . 

LEEUWENHOECK,*  two  hundred  years  ago,  recog- 
nised, and  described,  microscopic  organisms  in 
putrid  water  and  saliva,  which  probably  correspond 
with  organisms,  such  as  vibrios  and  leptothrix  ot 
modern  times.  During  two  centuries  these  minute 
beings  have  afforded  histologists  a  subject  for 
controversy  and  dispute.  Existing  as  they  do  upon 
the  very  borderland  of  the  vegetable  and  animal 
kingdoms,  not  only  have  they  been  transferred  from 
one  to  the  other,  but  even  the  question  has  been 
raised  whether  the  smaller  forms  should  be  con- 
sidered as  living  beings  at  all. 

In  reviewing  the  history  of  the  various  classifica- 
tions which  have  from  time  to  time  been  proposed, 
we  shall  see  that  the  gradual  improvements  in  the 
means  of  studying  such  minute  objects,  the  methods 
of  cultivating  them  artificially,  and  of  studying  their 
chemistry  and  physiology,  and  the  ever-increasing 
revelations  of  the  microscope,  have  resulted  in 

*  Leeuwenhoeck,  Oj>.  Omnia  (Lugd.  Batav.,  1722). 


1 76  BACTERIOLOGY. 

establishing  these  microscopic  objects  as  members 
of  the  vegetable  kingdom,  ranking  among  the 
lowest  forms  of  fungi.  While  enabling  us  to  settle 
their  position  as  a  whole,  these  improved  methods 
have  further  given  us  so  great  an  insight  into  the 
life-history  of  individual  forms,  that,  with  regard 
to  the  division  into  genera  and  species,  we  are 
up  to  the  present  time  still  in  a  position  of  doubt 
and  uncertainty. 

Miiller,  in  1773,  was  the  first  to  suggest  a  classi- 
fication. He  established  two  genera,  Monas  and 
Vibrio,  and  grouped  them  with  the  Infusoria.  In 
1824  Bory  de  Saint  Vincent  also  attempted  a  classi- 
fication ;  but  it  was  not  until  Ehrenberg  in  1838, 
and  Dujardin  in  1841,  worked  at  the  subject,  that 
a  scientific  distinction  of  species  was  attempted. 

Ehrenberg  described  four  genera  : — 

I.  Bacterium   .  .  filaments  straight,  rigid. 

II.  Vibrio          .  .  filaments  snake-like,  flexible. 

III.  Spirillum     .  .  filaments  spiral,  rigid. 

IV.  Spirochsete  .  .  filaments  spiral,  flexible. 

Dujardin  united  Spirillum  and  Spirochate,  and 
classed  them  thus  : — 

I.  Bacterium         .    filaments  rigid,  vacillating. 
II.  Vibrio       .         .    filaments  flexible,  undulatory. 
III.  Spirillum.         .    filaments  spiral,  rotatory. 

Up  to  that  time  bacteria  were  still  considered  as 


CLASSIFICATION    OF    BACTERIA.  177 

Infusoria;  but  the  year  1853  marked  the  com- 
mencement of  a  new  era  in  their  history,  for  Robin 
then  pointed  out  the  affinity  of  the  Bzcteria  and 
Vibrios  to  Leptothrix.  Davaine,  in  1859,  still 
more  definitely  insisted  that  the  Vibrios  were 
vegetables,  and  that  they  were  in  fact  allied  to 
the  Alga. 

Since  that  time  a  flood  of  light  has  poured  in 
upon  the  subject  through  the  writings  of  Hoff- 
mann, Pasteur,  Cohn,  Rabenhorst,  Hallier,  Billroth, 
Warming,  Nageli,  Magnin,  Marchand,  Sternberg, 
Van  Tieghem,  Lister,  Klein,  Koch,  Fliigge,  De 
Bary,  Zopf,  Cornil,  Babes,  and  many  other  workers 
in  the  recent  widespread  revival  of  bacteriological 
research. 

Of  all  these  writers  we  are  most  indebted  to 
Cohn,*  not  only  on  account  of  his  researches,  which 
extended  over  very  many  years,  but  also  for  his 
system  of  classification,  which  has  since  been 
almost  universally  adopted. 

In  his  first  classification,  published  in  1872,  Cohn 
considered  the  Bacteria  as  a  distinct  group  be- 
longing to  the  Algcej  and  divisible  into  four  tribes, 
including  six  genera  : — 

I.  Sphaerobacteria  globules  (Micrococcus), 

II.  Microbacteria  .  short  rods  (Bacterium). 

III.  Desmobacteria  .  long  rods  (Bacillus  and  Vibrio). 

IV.  Spirobacteria    .  spirals  (Spirochaete  and  Spirillum), 

*  Cohn,  Beitrdge  zur  Biologie  der  Pflanzen>  1872,  et  seq. 

12 


178  BACTERIOLOGY. 

Cohn  noted,  in  spite  of  placing  them  with  the 
Algce,  that  the  absence  of  chlorophyll  connected  the 
Bacteria  to  Fungi,  and  we  find  Nageli  subsequently 
adopting  this  view,  and  employing  the  term  Schi- 
zomycehs. 

Billroth,  in  1874,  disputed  the  division  into 
species,  and  considered  that  all  the  forms  described 
by  Cohn  were  but  developmental  forms  of  one  micro- 
organism, Coccobacteria  septica.  In  the  following 
year  Cohn  answered  the  criticism  of  Billroth,  and 
produced  a  second  classification,  in  which  he  still 
maintained  that  distinct  genera  and  species 
existed.  The  genera  Cohn  considered  to  be  dis- 
tinguished by  definite  differences  in  shape,  which 
were  adhered  to  throughout  life,  while  some  special 
feature,  as  a  difference  in  size  or  physiological 
action,  or  some  minute  difference  in  form,  deter- 
mined the  various  species.  Cohn  illustrated,  by  his 
well-known  comparison  of  a  sweet  and  a  bitter 
almond  the  appearances  of  which  are  similar  but 
the  properties  very  different,  that  a  distinction 
into  species  might  depend  upon  a  difference  in 
physiological  action  only.  Others  strongly  sup- 
port Cohn's  views.  By  cultivating  various  micro- 
organisms through  several  generations,  many 
conclude  that  a  micrococcus  cannot  be  trans- 
formed into  a  bacterium,  or  a  bacterium  into  a 
bacillus  or  spirillum.  Koch  does  not  believe—- 
and in  this  he  is  supported  by  Klein — that  a 
bacillus  can  change  its  nature,  and  be  converted 


CLASSIFICATION    OF    BACTERIA.  179 

from  a  harmless  into  a  pathogenic  form,  as  asserted 
by  Biichner.* 

The  second  classification  of  Cohn  (1875)  onty 
differed  from  the  first  in  that,  instead  of  keeping 
the  bacteria  as  a  separate  group,  he  placed  them, 
from  their  close  relationship  with  the  Phycochro- 
macea,  under  a  new  group,  the  Schizophytes,  and 
added  the  genera  Leptothrix,  Beggiatoa,  Crenothrix^ 
Sarcina,  Ascococcus,  Streptococcus ,  Myconostoc>  and 
Streptothrix. 

Nageli  maintained  that  Bacteria  were  allied  to 
Yeasts,  and  should  be  included  in  the  class  of  Fungi. 
In  fact,  he  divided  the  fungi  producing  decomposition 
into : — 

Mucorini          .  moulds 

Saccharomycetes    ....     yeasts 
Schizomycetes        ....     fission-fungi 

(This  last  class  comprising  bacteria.) 

Flugge,t  following  Rabenhorst,  maintained  the 
term  Schizomycetes,  and  divided  them  as  fol- 
lows : — 

*  Biichner,   Ueber  d.  exfierim.  Erzeugung  d.  Milzbrandconta- 
giums  aus  d.  Heupilzen. 

f  Fliigge,  Fermente  und  Mikrofiarasiten.     1883. 


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BACTERIOLOGY. 


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182  BACTERIOLOGY. 

The  belief  is  nevertheless  rapidly  gaining  ground 
that  the  lowest  forms  of  vegetable  life  cannot  be 
divided  by  a  hard  and  fast  line  into  a  series  with 
chlorophyll  (Alga),  and  a  series  without  it  (Fungi), 
and  the  tendency  now  is  to  solve  the  difference  of 
opinion  between  Cohn  and  Nageli  by  following  the 
example  of  Sachs,  and  amalgamating  the  two 
series  into  one  group,  the  Tkallophytes. 

Researches  by  competent  observers  have  quite 
recently  clearly  demonstrated  that  several  micro- 
organisms in  their  life  cycle  exhibit  successively 
the  shapes  characteristic  of  the  orders  of  Cohn. 

This  doctrine  of  pleomorphism,  now  widely  ac- 
cepted, was  distinctly  foreshadowed  in  a  publication* 
by  Lister  in  1873,  though  this  memoir  contained 
certain  conclusions  which  have  since  been  aban- 
doned. Lister  described  forms  of  cocci,  bacteria, 
bacilli,  and  streptothrix  in  milk,  which  he  regarded 
as  phases  of  the  same  micro-organism,  ^Bacterium 
lactis.  As  a  result  of  his  observations  Lister 
remarks  that  "  any  classification  of  bacteria  hitherto 
made  from  that  of  Ehrenberg  to  that  of  Cohn  based 
upon  absolute  morphological  characters  is  entirely 
untrustworthy."  To  Lankester,  however,  belongs 
the  credit  of  having  definitely  and  precisely  formu- 
lated this  doctrine.  In  a  paper, t  also  published  in 
1873,  Lankester  observed  that  the  series  of  form- 
phases  which  he  had  discovered  in  the  case  of  a 

*  Lister,  Quart.  Journ.  Microscofi.  Sci.,  1873,  pp.  380 — 408. 
t  Lankester,  Ibid,  pp.  408—425,  and  1876,  pp.  278—283. 


CLASSIFICATION    OF   BACTERIA.  183 

peach-coloured  bacterium  led  him  to  suppose  that 
the  natural  species  of  these  plants  were  "  within  the 
proper  limits  protean,  and  that  the  existence  of  true 
species  of  bacteria  must  be  characterised,  not  by 
the  simple  form-features  used  by  Cohn,  but  by  the 
ensemble  of  their  morphological  and  physiological 
properties  as  exhibited  in  their  complete  life- 
histories."  Lankester  inferred  that  these  phase- 
forms  were  genetically  connected  from  their  all 
possessing  the  common  characteristic  of  a  special 
pigment  bacterio-purpurin.  These  conclusions 
were  vigorously  opposed  by  Cohn,  and  doubt  still 
remains  in  the  minds  of  some  as  to  whether  the 
different  forms  are  really  only  stages  in  the  life- 
history  of  a  single  species.  Nevertheless  the 
theory  of  pleomorphism  has  steadily  gained  ground 
ever  since. 

Among  the  recent  observers  Cienkowski  and 
Neelsen  have  worked  out  the  different  forms  as- 
sumed by  the  bacillus  of  blue  milk ;  Zopf  has  in  a 
similar  manner  investigated  Clactotkrix,,  Beggiatoa, 
and  Crenothrix,  and  traced  out  various  forms  (Fig. 
66)  ;  Van  Tieghem  has  investigated  Bacillus  amylo- 
bacter  with  a  similar  result;  Hauser  has  quite 
recently  described  bacillar,  spirillar,  and  spirulinar, 
and  various  other  forms  in  the  Proteus  mnabilis  and 
Proteus  vulgans.  These  facts  obviously  shake  the 
very  foundation  of  Cohn's  classification,  and  we  are 
left  without  possessing  a  sound  basis  for  classifica- 
tion into  genera  or  species.  The  mode  of  repro- 


1 84 


BACTERIOLOGY. 


FIG.  66. 

CLADOTHRIX  DICHOTOMA— A.  Branched  Schizomycete :  (a)  Vibrio-form  ; 
(b)  Spirillum-form  [slightly  magnified].  B.  Screw-form  at  the  ends  : 
(a)  Spirillum-form ;  (b)  Vibrio-form.  C.  Very  long  Spirochaeta-form. 

D.  Branch  fragment,  at  one  end  Spirillum-form,  at  the  other  Vibrio-form. 

E.  Screw-form  :    (a)  Continuous  ;   (b)  Composed  of  rods,  and  (c)  Cocci. 

F.  Spirocheeta-form  :    (a)  Continuous  ;    (b}  Composed  of  long  rods,  (c) 
Short  rods,  and  (d)  Cocci  [after  Zopf]. 


CLASSIFICATION    OF    BACTERIA.  185 

duction  is  not  sufficiently  known  to  afford  a  better 
means  for  distinction  than  the  other  morphological 
appearances  taken  alone  ;  nor  can  we  depend  upon 
physiological  action,  which  is  held  by  many  to  vary 
with  the  change  of  form,  according  to  altered 
surroundings. 

Zopf,  who  has  warmly  supported  the  pleomor- 
phism  of  bacteria,  has  suggested  as  a  result  of 
his  investigations  a  division  of  the  Schizomycetes, 
Spaltpilze,  or  Fission-fungi,  into  the  following  four 
groups : — * 

1.  Coccacese. — Possessing  (so  far  as  our  knowledge 
at  present  reaches)  only  cocci,  and  thread  forms  resulting 
from  the  juxtaposition  of  cocci.     The  fission  occurs  in  one 
or  more  directions. 

Genera  : — Streptococcus,  Micrococcus,  Merismopedia, 
Sarcina,  Ascococcus. 

2.  Bacteriaceae. — Possessing    mostly    cocci,     rods 
(straight  or  bent),   and  thread-forms  (straight  or  spiral). 
The  first  may  be  absent,  and  the  last  possess  no  distinction 
between  base  and  apex. 

Division  (as  far  as  is  known)  occurs  only  in  one 
direction. 

Genera  ; — Bacterium,  Spirillum,  Vibrio,  Leuconostoc, 
Bacillus,  Clostridium. 

3.  Leptotricheae. — Possessing      cocci,      rods,     and 
thread-forms  (which  show  a  distinction  between  base  and 
apex).     The  last  straight  or  spiral. 

*  Zopf,  Die  S£altpilze,  1885. 


1 86  BACTERIOLOGY. 

Genera  : — Leptothrix,  Beggiatoa,  Crenothrix,  Phragmi- 
diothrix. 

4.   Cladotricheae. — Possessing  cocci,  rods,  threads, 
and  spirals.     Thread-forms  provided  with  false  branchings. 
Genus  : — Cladothrix. 

Zopf,  however,  does  not  assert  that  all  the  fission- 
fungi  exhibit  this  pleomorphism,  nor  does  he 
pretend  that  his  classification  will  include  all  the 
micro-organisms  described.  Cohn,  on  the  other 
hand,  was  ready  to  admit  that  all  the  forms 
described  by  him  were  not  truly  independent 
species.  Quite  recently  De  Bary,  Hueppe,  Baum- 
garten,  and  Fliigge  have  expressed  new  views  with 
regard  to  the  classification  of  bacteria. 

De  Bary  divides  them  into  two  great  groups — 
bacteria  which  form  endospores,  and  bacteria  which 
form  arthrospores.  This  affords  but  little  practical 
assistance,  though  regarded  by  botanists,  from  a 
scientific  standpoint,  as  a  step  in  the  right 
direction. 

Hueppe,  acknowledging  that  the  fructification 
must  eventually  be  made  the  basis  for  classification, 
suggests  an  arrangement  for  provisional  use  in 
which  this  view  is  introduced. 


CLASSIFICATION    OF    BACTERIA. 


187 


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1 88  BACTERIOLOGY. 

It  has  already  been  mentioned  that  the  produc- 
tion of  arthrospores  is  only  established  in  a  very 
few  species.  Therefore,  we  are  hardly  justified  in 
assuming  that  all  bacteria,  the  spore-formation  of 
which  is  quite  unknown,  are  to  be  included  with 
those  in  which  this  kind  of  fructification  has  been 
observed,  and  consequently  to  distinguish  genera 
on  the  same  grounds  may  be  considered,  to  say  the 
least,  somewhat  premature.  In  Baumgarten's  classi- 
fication the  genus  bacterium  is  dispensed  with,  and 
the  genera  divided  into  two  groups,  the  mono- 
rnorphic  and  the  pleomorphic. 

GROUP  I. — MONOMORPHIC. 
Genera . — Coccus. 
Bacillus. 
Spirillum. 

GROUP  II. — PLEOMORPHIC. 
Genera. — Spirulina. 
Leptothrix. 
Cladothrix. 

Fliigge  also,  in  his  recent  classification,  includes 
the  genus  bacterium  in  the  genus  bacillus.  The 
new  classification  differs  also  from  the  original  one 
in  the  grouping  together  of  the  different  species 
according  to  the  character  and  behaviour  of  the 
colonies  in  nutrient  gelatine.  The  abolition,  in 
Fliigge's  and  Baumgarten's  classification,  of  the 
genus  bacterium  is  no  doubt  owing  to  confusion 
having  arisen  from  the  distinction  between  a 


CLASSIFICATION    OF    BACTERIA.  189 

bacterium  and  a  bacillus,  being  a  question  of 
length.  Observers  differed  as  to  whether  a  rod 
of  a  certain  length  ought  to  be  considered  a  bac- 
terium or  a  bacillus.  To  meet  this  difficulty  a  rough- 
and-ready  rule  was  suggested,  viz.,  that  a  rod  less 
than  twice  its  breadth  in  length  should  be  considered 
as  a  bacterium,  and  otherwise  a  bacillus.  But  this 
purely  arbitrary  division  was  inadequate,  from  the  fact 
that  a  rod  at  one  stage  of  its  growth  or  under  certain 


FIG.  67. — BACTERIUM  PNEUMONIA  CROUPOSJE,  x  1500  (after  Zopf). 

conditions  might,  as  far  as  length  went,  truly  be  a 
bacterium,  and  under  other  circumstances  be  of 
such  a  length  as  to  entitle  its  being  considered  a 
bacillus.  We  avoid  such  confusion  if  we  follow 
Zopf,  and  acknowledge  as  a  difference  between  a 
bacterium  and  a  bacillus  the  presence  or  absence 
of  that  form  of  spore-formation  now  distinguished  as 
endogenous  spore-formation.  We  can  then  most 
conveniently  retain  this  generic  term,  to  include  that 
group  of  rod-forms  in  which  this  spore-formation  is 
as  yet  unknown  ;  moreover,  we  shall  find  that  by  so 


I QO  BACTERIOLOGY. 

doing,  with  one  or  two  exceptions,  we  get  collected 
together  those  short  rod-forms  (Fig.  67),  which 
appear  to  link  the  simple  cocci  to  the  spore-bearing 
rods  or  bacilli. 

This   must   surely   lead   to   less    confusion  than 


a 

FIG.  68. — EMMERICH'S  BACTERIUM,  x  700  (after  Emmerich). 

regarding  all  rod-forms  as  bacilli,  and  massing 
them  together  into  one  genus.  For  by  those  who 
adopt  the  latter  plan,  not  only  are  very  short  rods 
with  rounded  ends  included  as  bacilli,  e.g.,  Bacillus 


FIG.  69. — COLONIES  ON  NUTRIENT  GELATINE,    X  60. 

Neapolitans  or  Emmerich's  bacterium  (Fig.  68), 
but  even  cells  which  are  described  as  ovoid  are  also 
regarded  as  bacilli,  as  in  Loffler's  so-called  Bacillus 
parvus  ovatus* 


CLASSIFICATION    OF    BACTERIA.  IQI 

The  grouping  together  of  the  different  species 
according  to  the  character  of  the  colonies  in 
nutrient  gelatine  (Figs.  69,  70)  is  also  of  question- 
able advisability.  These  characters  can  hardly 


FIG.  70.— COLONIES  ON  NUTRIENT  AGAR-AGAR,  x  60. 

be  considered  to  be  of  sufficient  importance,  or 
indeed  in  many  cases  to  be  sufficiently  constant,  to 
serve  by  themselves  for  this  purpose.  In  many 
cases  a  slight  variation  in  the  composition  of  the 


FIG.  71.  -COLONY  OF  BACILLUS  ANTHRACIS,  x  60.    From  a  cover-glass 
impression-preparation,  stained  with  gentian-violet. 

nutrient  medium  may  considerably  affect  the  ap- 
pearances of  the  colonies.  At  the  same  time, 
the  appearances  are  very  characteristic  of  cer- 
tain species  of  bacteria  (Fig.  71),  and  in  other 


BACTERIOLOGY. 

cases  the  characters  of  the  colonies,  together  with 
the  characters  of  the  growths  in  test-tubes,  assist 
us  in  distinguishing  species  which  are  morpho- 
logically similar,  as  in  the  case  of  the  comma  bacilli 
of  Finkler  and  of  Koch. 

The  classification  here  given  will  be  found  to 
be  a  convenient  form  for  arranging  the  micro- 
organisms for  reference,  and  it  may  lead  the 
investigator  to  work  upon  the  same  lines  as  Zopf, 
and  by  tracing  the  life-history  of  individual  forms 
in  pure  cultivations,  either  to  extend  the  work  of 
establishing  protean  species  or  to  restrict  the 


FIG.  72.— BACTERIUM  OF  RABBIT  SEPTICAEMIA. 

doctrine  of  pleomorphism  to  a  very  few  forms. 
But  though  the  author  adheres  to  the  lines  of 
classification  proposed  by  Zopf,  he  is  not  prepared 
to  accept  his  teachings  in  their  entirety;  thus,  to 
embrace  all  described  species,  and  to  be  consist- 
ent with  the  author's  views,  it  has  been  necessary, 
not  only  to  add  to  Zopf  s  classification,  but  in  many 
cases  to  modify  his  arrangement  of  species.  For 
instance,  Zopf  regards  the  bacterium  of  rabbit 
septicaemia  (Fig.  72)  as  a  micrococcus,  and  the 
yellow  coccus  in  pus,  which  may  occur  in  short 
chains,  as  a  micrococcus.  Of  some  species  altera- 
tion in  the  nomenclature  is  justified  by  necessity. 


CLASSIFICATION    OF    BACTERIA.  193 

Thus  the  well-known  Micrococcus  projiigiosus,  now 
named  by  Fliigge  and  others  as  Bacillus  prodigiosus, 
is  placed  by  the  author  as  a  bacterium,  for  it  un- 
doubtedly appears  in  the  form  of  short  rods,  but 
without  endogenous  spore-formation. 

Any  arrangement  at  present  can  only  be  con- 
sidered provisional,  and,  therefore,  that  arrangement 
which  is  of  most  practical  assistance,  and  which  leads 
to  a  clear  description  of  the  important  characteristics 
on  which  the  final  classification  will  depend,  must 
be  considered  to  be  the  best.  For  example,  if  we 
abolish  the  genus  bacterium,  any  rod-form  may  be 
at  once  classed  as  a  bacillus  ;  on  the  other  hand, 
in  the  plan  here  adopted,  we  must  determine  the 
presence  or  absence  of  endogenous  spore-formation 
before  we  can  decide  whether  it  be  a  bacterium  or  a 
bacillus.  This  necessarily  leads  to  a  more  thorough 
study  of  their  life-history.  In  the  systematic  de- 
scription which  follows,  stress  is  laid  upon  the 
morphological  appearances  of  bacteria,  upon  the 
absence  or  presence  of  spore-formation,  and  upon 
the  appearances  under  cultivation,  in  addition  to 
other  characteristics,  such  as  the  changes  produced 
by  their  growth.  The  determination  of  species 
rests  upon  the  accumulated  evidence  afforded  by 
a  thorough  knowledge  of  their  life-history.  The 
form  of  the  organism,  the  changes  it  effects,  and 
the  microscopical  appearances  under  cultivation, 
must  be  collectively  taken  into  account. 


CHAPTER   XII. 

SYSTEMATIC  AND  DESCRIPTIVE. 

THE  Schizomycetes,  Spaltpilze,  or  Fission-fungi  have 
already  been  described  as  divisible,  according  to 
Zopf,  into  four  groups ;  '  Coccacece,  Bacteriacea,  Lep- 
totrichece,  and  Cladotrichecz .  They  comprise  the 
following  genera  and  species  : — 

GROUP  I. — COCCACECE. 

Genus  I.  Streptococcus  (Chain-cocci). — Division  in 
one  direction  only.  Individual  cocci  always  or 
occasionally  remain  united  together  to  form 
chains. 

Genus  II.  Merismopedia  (Plate-cocci). — Division  in 
two  directions,  forming  lamellae  or  plates. 

Genus  III.  Sarcina  (Packet-cocci). — Division  in  three 
directions,  forming  colonies  in  cubes  or  packets. 

Genus  IV.  Micrococcus  (Mass- cocci). — Division  in  one 
direction,  cocci  after  division  may  remain  aggre- 
gated in  irregular  heaps,  but  never  form  chains. 

Genus  V.  Ascococcus  (Pellicle-cocci). — Like  micro- 
coccus,  but  the  cocci  grow  in  characteristic 
gelatinous  pellicles. 


SYSTEMATIC    AND    DESCRIPTIVE. 


195 


Genus  I. — Streptococcus. 
SPECIES. 

ASSOCIATED  WITH  DISEASE  (many  Pathogenic  or  causally  related  to  disease]  : — 


In  man 


In  animals  \ 


/  Streptococcus  pyogenes 
Streptococcus  pyogenes  malignus 
Streptococcus  pyogenes  aureus 

Streptococcus  pyogenes  albus 
Streptococcus  pyogenes  citreus     . 
Streptococcus  cereus  albus   . 


Streptococcus  cereus  flavus  . 
Streptococcus  erysipelatosus 

vStreptococcus  toxicatus 
Streptococcus  in  puerperal  fever  . 
Streptococcus  in  endocarditis 
Streptococcus  in  diphtheria  . 
Streptococcus  articulorum     . 

Streptococcus    in     cerebro-spinal 
meningitis         .... 
Streptococcus  in  yellow  fever 
Streptococcus  in  dental  caries 
Streptococcus  varioloe  et  vacciniae 

Streptococcus  of  swine-erysipelas 
Streptococcus  of  cattle-plague 
Streptococcus  of  loot  and  mouth 

disease      

Streptococcus  of  septicaemia  con- 
secutive to  anthrax    . 
Streptococcus  septicus . 
Streptococcus  of  progressive  tissue 
necrosis  in  mice 


Streptococcus  perniciosus     . 
Streptococcus  bombycis 
\  Streptococcus  insectorum 

UNASSOCIATED  WITH  DISEASE  : — 
Streptococcus  viscosus . 
Streptococcus  coronatus 
Streptococcus  radiatus 
Streptococcus  flavus  desidens 


Pathogenic  in  man  and  ani- 
mals. 

Saprophytic  in  man,  patho- 
genic in  animals. 

Pathogenic  in  man  and  ani- 
mals. 


Associated  with  pus,  but  not 
pathogenic  in  man  or  ani- 
mals. 

"  » 

Pathogenic  in  man  and  ani- 
mals. 

Pathogenic  (?). 
Possibly  saprophytic  only. 


Saprophytic   in  man,   patho- 
genic in  animals. 

Possibly  saprophytic  only. 


Pathogenic   in  man   (?)   and 

animals  (?). 
Pathogenic  (?). 
Pathogenic. 


Pathogenic  (?). 

Pathogenic. 

Possibly  saprophytic  only. 


Zymogenic  saprophyte 
Simple  saprophyte. 


Streptococcus  pyogenes,  Rosenbach  (Chain 
micrococci  in  pus:  Coccus  of  Py&mia).  —  Cocci 
occurring  singly,  in  chains,  and  in  zooglcea.  Inocu- 


1 96  BACTERIOLOGY. 

lation  of  guinea-pigs  or  mice  with  pus  containing 
these  cocci,  or  with  a  pure  cultivation  of  the  same, 
causes  suppuration  at  the  site  of  injection,  and 
death  with  symptoms  of  blood-poisoning.  In  the 
blood,  in  the  tissue  around  the  abscess,  and  in 
the  pus  the  cocci  are  found  singly  or  in  zooglcea 
or  chains.  Their  appearances  in  cultivation-media 
have  been  very  minutely  described.*  Cultivated 
in  a  streak  on  the  surface  of  nutrient  gelatine  on 
a  glass  plate,  they  form  at  h'rst  whitish,  somewhat 
transparent,  rounded  spots,  of  the  size  of  small 
grains  of  sand.  They  develop  but  slightly  on  this 
medium,  even  at  the  highest  temperature  attain- 
able without  liquefying  the  gelatine.  On  nutrient 
agar-agar  they  grow  most  energetically  at  a 
temperature  of  35° — 37°C.  On  this  medium  also, 
they  show  a  tendency  to  form  little  spots,  which 
finally  become  about  the  size  of  a  pin's  head. 
If  a  streak  is  made  with  a  needle  well  charged 
with  a  fresh  cultivation,  growth  in  a  continuous 
line  is  obtained,  but  still  showing  an  inclination  to 
form  centres.  In  its  further  development  the 
middle  of  the  cultivation  is  heaped  up,  and  presents 
a  pale  brownish  coloration,  while  the  periphery  is 
flattened,  except  at  the  extreme  margin,  which  is 
again  raised  up,  and  often  with  a  spotted  appear- 
ance. Still  later,  the  periphery  develops  successive 
layers  or  terraces.  The  growth  is  so  slow  that 

*  Rosenbach,    Mikro-organismen    bet    den     VFund- Infection s- 
Krankheiten  des  Menschen.     1884. 


SYSTEMATIC    AND    DESCRIPTIVE.  197 

in  two  to  three  weeks  the  maximum  width  of  the 
culture-streak  is  about  2  to  3  mm.  On  solidified 
blood  serum  the  cocci  grow  as  on  agar-agar. 
They  do  not  liquefy  any  nutrient  medium.  In 
a  vacuum  they  rapidly  cause  the  decomposition 
of  white  of  egg  or  beef,  which  are  energetically 
peptonised.  They  occur  in  acute  abscesses.* 

Streptococcus  pyogenes  malignus.  Fliigge. 
— Cocci  occurring  singly  and  in  chains.  They 
grow  very  slowly  in  nutrient  gelatine  ;  the  cultiva- 
tions closely  resemble  those  of  Streptococcus  pyogenes. 
They  are  pathogenic  in  mice  and  rabbits.  They 
were  isolated  from  necrotic  patches  in  the  spleen 
of  a  fatal  case  of  leukaemia. 

Streptococcus  pyogenes  aureusf  {Staphylo- 
coccus* pyogenes  aureus,  Rosenbach.  Yellow  coccus  in 
pus.  Coccus  of  acute  infectious  osteomyelitis). — Cocci 
singly,  in  pairs,  short  chains,  and  irregular  masses. 
Cultivated  on  nutrient  agar-agar  an  orange-yellow 
culture  develops,  looking  like  a  streak  made  with 
oil  paint  \  (Plate  VIII.,  Fig.  2).  Cultivated  in  a 
test-tube  of  nutrient  gelatine,  the  gelatine  is  rapidly 
liquefied,  and  the  growth  subsides  as  an  orange- 

*  Ogston,  Brit.  Med.  Journ.     1881. 

t  These  cocci  are  placed  among  the  Streptococci  as  they  un- 
doubtedly may  occur  in  small  chains  of  three  or  four  individuals 
linked  together.  The  form  of  zooglcea  is  not  considered  important. 
The  same  applies  to  the  Staphylococcus  cereus  albus  and  flavus, 
which  are  also  placed  by  the  author  in  the  genus  Streptococcus. 
Passet  acknowledges  that  they  form  long  and  short  chains,  but 
named  them  Staphylococcus  because  of  the  prevalence  of  irregular 
zooglcea. 

Rosenbach. 


I  9  8  BACTERIOLOGY. 

yellow  sediment.  On  potatoes  and  blood  serum  a 
similar  orange-yellow  culture  grows  luxuriantly. 

The  micro-organisms  injected  into  the  pleura  or 
knee  of  a  rabbit  produce,  as  a  rule,  a  fatal  result 
on  the  following  day,  but  if  it  survives  longer,  it 
eventually  dies  of  severe  phlegmon.  If  injected 
into  the  knee  of  a  dog,  suppuration  occurs,  fol- 
lowed by  disintegration  of  the  joint.  The  cocci  do 
not  cause  any  septic  odour  in  pus,  nor  does  any 
gas  develop.  Albumen  is  converted  by  their  action 
into  peptones. 

They  occur  in  the  pus  of  boils  and  in  the 
abscesses  of  pyaemia,  puerperal  fever,  and  acute 
osteomyelitis.  Injected  into  the  peritoneal  cavity 
of  animals,  they  set  up  peritonitis,  and  introduced 
into  the  jugular  vein  they  produce  septicaemia  and 
death.  When  a  small  quantity  of  a  cultivation  was 
introduced  into  the  jugular  vein  after  previous 
fracture  or  contusion  of  the  bones  of  the  leg,  the 
animal  died  in  about  ten  days,  and  abscesses  were 
found  in  and  around  the  bones,  and  in  some  cases 
in  the  lungs  and  kidneys.  Similar  cocci  were  found 
in  the  blood  and  pus  of  the  animals.* 

Streptococcus  pyogenes  albus  (Staphyio- 
coccus  pyogenes  albus,  Rosenbach).  Cocci  micro- 
scopically indistinguishable  from  the  above.  In 
cultivations  also  they  resemble  the  Streptococcus 
pyogenes  aureus,  but  the  growth  consists  of  opaque 
white  masses.  They  liquefy  nutrient  gelatine 

*  Becker,  Deutsche  Med.  Wochenschr.     Nov.  1883. 


SYSTEMATIC    AND    DESCRIPTIVE.  IQQ 

rapidly,  and  subside  to  the  bottom  as  a  white 
sediment.  They  are  also  similar  to  the  above- 
mentioned  in  their  pathogenic  action.  Pure 
cultivations  of  the  organism  were  obtained 
from  a  case  of  acute  suppuration  of  the  knee- 
joint. 

Streptococcus  pyogenes  citreus  (Staphylo- 
coccus  pyogenes  citreus*  Passet).  Cocci  singly,  in 
pairs,  chains,  and  irregular  masses.  If  cultivated 
on  nutrient  gelatine  or  nutrient  agar-agar,  a  sulphur 
or  lemon-yellow  growth  develops  (Plate  XXIV., 
Fig.  3).  When  inoculated  under  the  skin  of  mice, 
guinea-pigs,  or  rabbits,  an  abscess  forms  after  a 
few  days,  from  which  a  fresh  cultivation  of  the 
micro-organism  can  be  obtained.  They  are 
frequently  present  in  pus. 

Streptococcus  cereus  albus  (Stapkylococcus 
cereus  albus,  Passet).*  Cocci,  morphologically  simi- 
lar to  the  above,  but  distinguished  by  forming  on 
nutrient  gelatine  a  white,  slightly  shining  layer, 
like  drops  of  stearine  or  wax,  with  somewhat 
thickened,  irregular  edge.  The  needle  track 
develops  into  a  greyish-white,  granular  thread. 
In  plate-cultivations,  on  the  first  day,  white  points 
are  observed,  which  spread  themselves  out  on  the 
surface  to  spots  of  i — 2  mm.  When  cultivated 
on  blood-serum  a  greyish-white,  slightly  shining 
streak  develops,  and  on  potatoes  the  cocci  form 
a  layer  which  is  similarly  coloured. 

*  Passet,  Fortschritte  der  Medicin,  Jan.  i5th  and  Feb.  ist,  1885. 


200  BACTERIOLOGY. 

Streptococcus  cereus  flavus  (Staphylococcus 
cereus  flavus,  Passet).* — Cocci  which  also  occur  in 
pus.  If  cultivated  in  nutrient  jelly  the  growth,  which 
is  at  first  white,  becomes  lemon-yellow,  somewhat 
darker  in  colour  than  Streptococcus  pyogenes  citreus. 
Microscopically  Streptococcus  cereus  flavus  corre- 
sponds with  Streptococcus  cereus  atbus,  and  they  both 
form  zooglcea  of  medium-sized  cocci  (diam.  1*16/1). 
Inoculation  experiments  with  both  kinds  gave  nega- 
tive results.  Among  the  micro-organisms  present 
in  pus  a  coccus  has  been  described  as  occurring  oc- 
casionally, which  is  almost  identical  with  Bacterium 
pneumonia  crouposce ;  compare  also  Streptococcus 
pyogenes  (p.  195)  and  Streptococcus  pyogenes  aureus 
(p.  197). 

Streptococcus  erysipelatosus. — Minute  cocci 
-4  p,  to  *3  p.  occur  in  chains  in  human  erysipelatous 
skin,  and  in  the  fluid  of  erysipelatous  bullse.  They 
occupy  the  lymphatic  channels  of  the  skin,  and 
spread  along  them  as  the  disease  progresses. f 

They  can  be  cultivated  artificially  in  nutrient  gela- 
tine or  agar-agar,  and  produce  typical  erysipelas 
when  re-inoculated  in  man  or  animals. J  The 
characteristic  erysipelatous  blush  is  produced  by 
inoculating  these  micro-organisms  in  the  ear  of 
a  rabbit.  In  the  human  subject  the  disease  was 
produced  in  fifteen  to  sixty  hours  after  inoculation. 

*  Passet,  ibid. 

t  Lukomsky,  Virch.  Archiv,  vol.  Ix. 

\  Orth,  Archiv  fur  Exp.  Pathol.  u.  Pharmacol.,  Bd.  i.  1873. 


SYSTEMATIC    AND    DESCRIPTIVE.  2OI 

A  beneficial  result  was  obtained  in  cases  of  lupus, 
cancer,  and  sarcoma,  this  being  the  object  for 
which  the  latter  inoculations  were  undertaken.* 

The  appearances  of  cultivations  very  strongly 
resemble  those  already  described  in  Streptococcus 
pyogenes.  There  is  less  tendency,  however,  to  the 
formation  of  terraces,  the  edge  of  the  growth  is 
thicker  and  more  irregular,  and  the  appearance  of 
the  streak  is  more  opaque  and  whiter.f 

Streptococcus  toxicatus,  Burrill. — Globular 
cells,  *5  p,  in  diam,,  singly,  in  pairs,  and  rarely  in 
chains.  They  occur  in  species  of  Rhm  (SumacJi), 
and  have  been  credited  with  being  the  cause  of  the 
inflammation  which  is  produced  by  the  poison  of 
the  plant. 

Streptococcus  in  puerperal  fever.J — Cocci  in 
zoogloea,  and  sometimes  in  chains,  have  been  ob- 
served in  all  organs  affected  in  puerperal  fever,  and 
especially  in  the  endocardium,  lung,  spleen,  kidney, 
and  brain. 

Streptococcus  in  endocarditis,  Klebs — Cocci 
i  p,  and  '5  /*  in  diam.,  and  chains.  They  have  been 
observed  in  masses  upon  the  altered  valves  and  in 
the  detritus  of  the  ulcerations  of  the  endocardium 

*  Fehleisen,  Aetiologie  des  Erysipels.     1883. 

t  Rosenbach. 

J  In  this  and  many  similar  cases  the  cocci  are  given  as  distinct 
species  from  their  association  with  particular  diseases,  not  because 
they  are  believed  to  be  causally  related,  for  there  is  very  little 
evidence  in  favour  of  that  belief  as  yet,  but  purely  for  convenience 
of  reference.  In  many  cases  they  are  probably  only  septic  organisms, 
which  have  found  a  pabulum  in  the  dead  tissue  ;  others  appear  to  be 
identical  with  organisms  which  have  been  found  in  pus. 


?O2  BACTERIOLOGY. 

in  endocarditis  ulcerosa  ;  as  chains  also  in  the  muscle 
of  the  heart,  and  forming  plugs  in  the  vessels  of 
the  heart,  spleen,  and  kidney.  Some  forms  are 
identical  with  Staphylococcus  pyogenes  aureus* 
Micrococci  have  also  been  described  in  connection 
with  chronic  and  other  forms  of  endocarditis. 

Streptococcus  in  diphtheria. — Oval  cocci, 
•35  to  ri  \L  in  diameter,  have  been  described  as 
characteristic  of  diphtheria. t  The  cells  lie  singly, 
in  pairs  or  in  rosaries,  and  in  spherical  or  cylin- 
drical masses  in  diphtheritic  membranes  and  the 
surrounding  connective  and  muscular  tissues. 
From  the  point  of  infection  they  can  be  traced 
along  the  lymphatics,  and  are  found  in  the  blood, 
heart,  liver,  kidneys,  and  other  organs.  In  severe 
cases  the  blood  capillaries  and  uriniferous  tubules 
are  blocked  up. 

The  attempt  to  get  pure  cultivations  in  nutrient 
media  and  to  make  inoculation  experiments  has 
not  yet  succeeded  ;  a  bacterium  has  also  been 
described  as  associated  with  diphtheria  (p.  230). 

Streptococcus  articulorum,  Loffler. — Cocci 
forming  remarkably  long  chains.  Cultivated  in 
nutrient  gelatine  they  form  pale-grey  watery- 
looking  droplets.  They  frequently  produce  a 
fatal  result  when  inoculated  in  mice,  and  the 
chains  are  found  in  the  spleen  and  other  organs. 
Rabbits  inoculated  subcutaneously  in  the  ear 

*  Ziegler,  Patholog.  Anatomy.     1885. 

t  Oertel,  Deutches  A  rchivf.  Klin.  ^Med.     1871. 


SYSTEMATIC    AND    DESCRIPTIVE.  2OJ 

suffered  from  an  erysipelatous  inflammation.  In- 
jection of  the  cocci  into  the  circulation  produced 
suppurative  inflammation  of  the  joints.  Isolated 
from  the  mucous  membrane  of  diphtheritic  cases. 

Streptococcus  in  cerebro-spinal  meningitis.* 
— Cocci,  diplococci,  and  chains  have  been  observed 
in  the  exudation  of  cerebro-spinal  meningitis  ;  all 
forms  were  detected  in  the  meninges,  and  zoogloea 
in  the  kidneys. 

Streptococcus  in  yellow  fever — Cocci  '6-7  ^ 
in  diameter  have  been  observed  in  this  disease. f 
They  occur  in  chains,  aggregated  in  masses,  which 
distend  the  vessels  of  the  kidney  and  liver. 

Streptococcus  in  dental  caries. — Two  species 
of  streptococci  are  believed  to  be  intimately  con- 
nected with  caries  of  the  teeth.  J 

1.  Occurs  in  the  form  of  cocci,  diplococci,  and 
chains,    which    develop    very   rapidly    in    nutrient 
gelatine,  speedily  converting  it  into  a  turbid  liquid. 
They  are  agents  of  lactic  acid  fermentation. 

2.  Occurs  as  very  small  cocci,  rarely  in  chains, 
which  rapidly  liquefy  nutrient  gelatine.     There  are 
also  associated  with  these,  two  species  of  micrococci 
and  a  spirillum  (Spirillum  sputigenuni). 

Streptococcus  variolae  et  vacciniae. — Cocci, 
•5  ju,  in  diam.,  singly,  in  pairs,  and  in  long  or  short 
chains,  and  colonies.  They  are  found  in  the  fresh 


*  Leyden,  Central  Blatt f.  Klin.  Med.     1883. 
t  Cornil  and  Babes,  Les  Bacteries.     1885. 
1  Miller,  Deutsche  Med.  Woch.     1884. 


204  BACTERIOLOGY. 

lymph  *  of  human  and  cow-pox,  and  in  the  pustules 
of  true  small-pox.  They  are  regarded  as  the  active 
principle  of  vaccine  lymph,  since  filtration  deprives 
the  latter  of  its  infectious  element. f  The  lympha- 
tics of  the  skin  in  the  region  of  the  pustule  of  both 
human  and  sheep-pox  are  filled  with  cocci.  Suc- 
cessful vaccination  has  been  stated  to  result  from 
artificial  cultivations. J 

Streptococcus  of  swine-erysipelas  (Microbe  du 
r ou get du pore,  Pasteur). — Cocci, §  diplococci,  chains, 
and  zooglcea  have  been  described  as  present  in  the 
blood  in  "  rouget  du  pore"'  The  microbes  have  no 
effect  upon  fowls,  but  kill  rabbits  and  sheep.  In- 
oculated into  healthy  pigs  they  give  rise  to  the 
disease,  and  occasion  a  fatal  result.  Inoculation 
with  weakened  virus  protects  against  virulent  matter. 

Streptococcus  of  cattle-plague,  Semmer. — 
Cocci  occurring  singly  in  chains  and  zooglcea. 
They  grow  rapidly  in  artificial  media.  A  calf 
inoculated  from  a  cultivation  died  in  seven  days 
from  cattle-plague.  The  cocci  lose  their  virulence 
bv  successive  cultivation,  and  the  weakened  cultiva- 
tions protect  against  the  virulent  disease.  They 
were  observed  in  the  blood  and  lymphatic  glands, 
and  cultivated  from  the  latter. 

*  Cohn,  Virchow's  Archiv.     1872. 

t  Chauveau,  Cumptes  Rendus.  1868.  Burdon  Sanderson,  Reports 
on  the  intimate  pathology  of  contagion. 

\  Quist,  St.  Petersburg^  Med.  Wochenschrift.     1883. 

§  According  to  Loffier  these  cocci  are  associated  with  a  minute 
bacillus  which  is  the  true  cause  of  the  disease  (vide  Bacillus  of 
swine-erysipelas). 


SYSTEMATIC    AND    DESCRIPTIVE.  2O5 

Streptococcus  of  foot  and   mouth  disease, 

(Microcoaus  of  foot  and  mouth  disease,  Klein). — Cocci 
singly,  in  dumb-bells,  and  in  curved  chains.  They 
grow  well  in  milk,  in  alkaline  peptone- broth,  in 
nutrient  gelatine,  and  in  nutrient  agar-agar. 
Cultivated  on  obliquely  solidified  nutrient  gelatine 
they  form  a  film  composed  of  minute  granules  or 
droplets  closely  placed  side  by  side,  but  not  con- 
fluent. The  gelatine  is  not  liquefied.  Observed 
in  the  vesicles  of  sheep  suffering  from  foot  and 
mouth  disease. 

Streptococcus  of  septicaemia  consecutive 
to  anthrax,  Charvin. — In  rabbits,  some  hours  after 
death  from  anthrax,  cocci  in  chains  have  been 
found  in  the  kidney  and  elsewhere.  Inoculated 
into  rabbits  fatal  septicaemia  was  produced  without 
the  presence  of  suppuration. 

Streptococcus  septicus,  Nicolaier  — Cocci, 
diplococci,  and  chains.  They  grow  very  slowly  in 
nutrient  media,  forming  in  plate-cultivations  after 
several  days  minute  dot-like  colonies.  In  mice 
they  produce  paralysis  of  the  hind  extremities  and 
death,  and  in  rabbits  a  local  inflammation  and 
death  after  two  or  three  days.  Isolated  from  earth. 

Streptococcus  of  Progressive  Tissue  Ne- 
crosis in  Mice.* — Cocci  "5  p,  in  diam.,  in  chains 
or  rosaries,  and  zoogloea ;  their  invasion  causes 
tissue  necrosis  with  destruction  even  of  cartilage 

*  Koch,.  Untersuch.  iiber  die^Etiologied.  Wundinfections  Krank- 
heitcn.     1878.     [New  Syd.  Soc.] 


205 


BACTERIOLOGY. 


cells,  and  spreading  from  the  point  of  inoculation, 
causes  death  in  about  three  days  (Fig.  73).  The 
cocci  are  absent  from  the  blood  and  internal 
organs.  These  observations  were  made  after  the 
injection  of  putrid  fluids  in  the  ear  of  mice,  and 
a  pure  infection  was  obtained  by  the  inoculation 
of  field-mice,  which  have  an  immunity  from  bacil- 
lary  septicaemia. 

''  ':    "',V 

Q  •   if'tf^"" 

rS !  I    •  I  ! 


'^3* 
fctf> 


FIG.  73.  —STREPTOCOCCUS  OF  PROGRESSIVE  TISSUE  NECROSIS  IN  MICE. 
(a)  Necrotic  cartilage  cells,  and  (£)  Chains  in  masses  ;  (<:)  Chains 
isolated  [after  Koch]. 

Streptococcus  perniciosus  (Parrot  disease]. 
—Cocci,  singly,  in  chains,  and  in  zooglcea  have 
been  described  in  connection  with  a  disease  of  the 
grey  parrot  (Psittacus  erithacus).*  This  disease 
is  fatal  to  about  80  per  cent,  of  these  parrots 
imported  to  Europe.  They  suffer  from  diarrhoea 
and  general  weakness  ;  their  feathers  are  ruffled, 
their  wings  hang  loosely,  and  their  eyelids  close  ; 
convulsions  set  in,  and  death  follows.  At  the 


*  Wolff,  Virchouts  Archtv.     1883. 


SYSTEMATIC    AND    DESCRIPTIVE:.  2OJ 

autopsy  greyish  nodules  are  found  in  the  lungs, 
liver,  spleen,  and  kidney ;  in  and  around  the 
capillaries  of  these  nodules,  and  in  the  blood  of  the 
heart;  the  cocci  are  found  in  great  numbers  in 
zooglcea,  and  more  rarely  in  chains.  Inflammatory 
change  in  the  surrounding  tissue  is  absent. 

Streptococcus  bombycis,  Bechamp  (Micro- 
zyma  bombycis). — Oval  cocci  "5  p,  in  diam.,  singly, 
in  pairs,  and  chains.  They  occur  in  the  contents 
of  the  alimentary  canal,  and  in  the  gastric  juice  of 
silkworms  suffering  from  " flacherU"  ("  Maladie  de 
mortsblancs"  " flaccidczza"  "  scklafsucht"). 

Streptococcus  insectorum,  Burrill. — Obtusely 
oval  cells,  7 — i  p,  long  and  -55  p  broad,  singly, 
in  pairs,  chains,  or  zoogloea.  They  were  detected 
in  the  digestive  organs  of  the  chinck-bug  (Blissus 
leucopterus)  when  suffering  from  a  certain  contagious 
disease. 

Streptococcus  viscosus,  Pasteur. — Globular 
cells  *2  ju,  in  diam.,  singly  or  in  chains.  These  and 
allied  forms  have  been  considered  to  be  the  cause 
of  mucoid  fermentation  in  wine  and  beer  *  (vin 
filant,  biere  ma  lade). 

Streptococcus  coronatus  (Micrococcus  corona- 
tus,  Fliigge). — Cocci  i  //,  in  diam.,  singly,  in  short 
chains,  and  in  zooglcea.  In  plate-cultivations  the 
colonies  have  a  characteristic  halo,  formed  by  the 
liquefaction  of  the  gelatine  around  the  colony. 
Isolated  from  the  air. 

*  Pasteur,  Etudes  sur  le  Vin  ;  sur  la  Biere.     1866  ;   1876. 


2O3  BACTERIOLOGY. 

Streptococcus  radiatus  (Micrococcus  radialus, 
Fliigge). — Cocci  less  than  i  p,  in  diam.,  singly,  and 
in  short  chains.  They  rapidly  form  whitish  colonies 
with  a  yellowish-green  sheen.  They  liquefy  the 
gelatine,  the  colonies  sinking  down,  and  after  one 
or  two  days  developing  a  circlet  of  rays.  A 
peculiar  ray-like  appearance  is  characteristic  also 
of  the  growth  in  test-tubes.  Isolated  from  con- 
taminated plate-cultivations. 

Streptococcus  flavus  desidens  (Micrococcus 
flavus  desidens,  Fliigge). — Cocci,  diplococci,  and 
short  chains.  They  form  yellowish-white  colonies, 
which  gradually  sink  down  in  the  gelatine.  In  test- 
tubes  they  form  china-white,  confluent  masses  in 
the  track  of  the  needle,  and  on  the  surface  a 
yellowish-brown  slimy  layer.  Isolated  from  con- 
taminated plate-cultivations. 


Genus  II. — -Merhmopedia. 
SPECIES. 

ASSOCIATED  WITH  DISEASE  :— 

Merismopedia  gonorrhoeas   .         .    Pathogenic  in  man. 

Micrococcus  tetragon.       .        i  jJ^ggf 
Diplococcus  albicans  tardissimus     Saprophytic  in  man. 
UNASSOCIATED  WITH  DISEASE  : — 

Micrococcuscitreus  conglomerates     Simple  Saprophyte. 
Micrococcus  subflavus  ,,  ,, 

Micrococcus  albicans  amplus        .          „  ., 

Merismopedia     Gonorrhoeae     (Coccus    of 
Gonorrhoea). — Cocci    0*83    p,    in    diam.,    singly,    in 
pairs,  in  tetrads,  and  zooglcea  groups.     They  are 


SYSTEMATIC    AND    DESCRIPTIVE.  2OQ 

found  in  gonorrhceal  pus  adhering  to  the  pus 
corpuscles  and  epithelial  scales.  Artificial  cultiva- 
tions have  been  carried  out,*  and  the  pathogenic 
character  of  the  cocci  established  by  inoculation. 

Micrococcus  tetragonus.— Cocci  about  i  /x 
in  diam.,  in  groups  of  four  (tetrads),  surrounded 
by  a  hyaline  capsule.  They  are  found  in  the 
sputa  of  phthisical  patients  and  in  the  walls  of 
tubercular  cavities.  In  a  test-tube  of  nutrient 
gelatine  they  form  an  irregular  white  growth,  more 
especially  in  the  upper  part  of  the  needle  track 
(Plate  IV.,  Fig.  i).  On  the  sloping  surface  of 
nutrient  agar-agar  thick,  whitish,  heaped-up  masses 
develop.  Guinea-pigs  and  mice  inoculated  with  a 
minute  quantity  of  a  pure  cultivation  die  in  two 
to  ten  days,  and  the  groups  of  the  characteristic 
tetrads  may  be  found  in  the  capillaries  through- 
out the  body,  especially  in  the  spleen,  lung,  and 
kidney  (Plate  XII.,  Fig.  i). 

Double  infection  can  be  produced  by  inoculating 
a  mouse  with  a  pure  cultivation  of  Bacillus 
anthracis  two  or  three  days  after  inoculation 
with  Micrococcus  tetragonus.  On  examination  after 
death  the  capillaries  of  the  lungs,  liver,  and 
kidney  are  filled  with  both  anthrax  bacilli  and 
masses  of  tetrads  t  (Plate  XVII.,  Fig.  2). 

Micrococcus  citreus  conglomeratus,  Bumm. 

*  Bockhart,  Sitzungsberichte  der  Phys.  Med.  Gesell.  Wiirzburg. 
1882. 
tThe  Author,  "  Notes  from  a  Bacteriolog.  Labomtory,"  Lancet, 

1885. 


210  BACTERIOLOGY. 

— Cocci  1*5  /A  in  diam.,  similar  to  gonococci. 
They  form  lemon-yellow  colonies  on  plate-cultiva- 
tions. Isolated  from  blennorrhceic  pus  and  from 
dust  from  the  air. 

Diplococcus  albicans  tardissimus,  Bumm. — 
Cocci  morphologically  identical  with  gonococci. 
They  grow  extraordinarily  slowly  on  gelatine. 
They  were  isolated  from  urethral  pus. 

Micrococcus  subflavus,  Bumm. — Cocci  mor- 
phologically resembling  gonococci.  Cultivated  on 
nutrient  gelatine,  they  form  whitish  dots  which 
become  gradually  greyish  and  then  yellow  in 
colour,  and  confluent.  They  were  observed  in 
lochial  discharges  and  vaginal  secretions. 

Micrococcus  albicans  amplus,  Bumm.  — 
Cocci  in  pairs  and  tetrads  similar  to  gonococci, 
but  considerably  larger.  Found  in  vaginal  secre- 
tions. 

Genus  III. — Sarcina. 

SPECIES. 
UNASSOCIATED  WITH  DISEASE  : — 

l±™tiaca         :}chro™ogenic  saprophytes. 

Sarcina  ventriculi 

Sarcina  intestinalis 

Sarcina  urinse 

Sarcina  litoralis     .         .  \  Simple  saprophytes. 

Sarcina  Reitenbachii 

Sarcina  hyalina 

Sarcina  alba          + 

Sarcina  lutea. — Cocci  singly,  in  pairs,  in 
tetrads,  and  in  packets.  A  single  individual  in  a 
tetrad  may  be  divided  into  two,  or  into  four,  so 
that  a  tetrad  within  a  tetrad  results.  Cultivated 


SYSTEMATIC    AND    DESCRIPTIVE.  211 

in  nutrient  agar-a.gar  in  a  test-tube,  they  form  a 
colourless  growth  along  the  track  of  the  needle, 
and  a  bright  canary-yellow  layer  upon  the  surface, 
v/here  they  have  access  to  the  air  (Plate  XIII., 
Fig.  i,  Plate  VIII.,  Fig.  i).  In  plate-cultivations  the 
colonies  are  round,  slightly  granular  in  appearance, 
and  yellow.  Cultivated  in  a  test-tube  containing 
nutrient  gelatine,  they  grow  rapidly ;  the  gelatine 
becoming  liquid,  the  yellow  growth  forms  a  wad 
about  the  middle  of  the  tube  (Plate  V.,  Fig.  2),  or, 
liquefying  the  whole  of  the  gelatine,  subsides  to 
the  bottom  of  the  test-tube.  Cultivated  on  sterilised 
potatoes  they  form  a  yellow  layer  (Plate  X.,  Fig  i). 
In  drop-cultures  in  bouillon  the  subdivision  into 
tetrads  within  tetrads  and  formation  of  groups  of 
8,  1 6,  and  24  can  be  studied  (Plate  L,  Fig.  7).  In- 
oculation of  mice  produces  negative  results.  The 
cocci  are  occasionally  present  in  the  air. 

Sarcina  aurantiaca. — Cocci  singly,  in  pairs, 
in  tetrads,  and  in  packets.  They  form  small 
orange-yellow  colonies  on  plate-cultivations,  and 
in  test-tubes  slowly  liquefy  the  gelatine  along  the 
whole  needle  track,  forming  on  the  surface  an 
orange-yellow  growth.  On  potatoes  they  slowly 
develop  the  same  pigment. 

Sarcina  ventriculi,  Goodsir.* — Cocci  reach- 
ing 4  //,  in  diam.,  united  in  groups  of  four,  or 
multiples  of  four,  producing  cubes  or  packets  with 
rounded-off  corners.  Contents  of  the  cells  are 

*  Goodsir,  Edinburgh  Med.  and  Surg.  Journal.     1842. 


2  I  2  BACTERIOLOGY. 

greenish  or  yellowish-red.  They  occur  in  the 
stomach  of  man  and  animals  in  health  and  disease, 
and  were  first  detected  in  vomit. 

Sarcina  intestinalis,  Zopf.* — Cocci  in  groups 
of  four  or  eight.  Very  regular  in  form ;  never  in 
the  large  packets  which  occur  in  Sarcina  ventriculi. 
They  are  found  in  the  intestinal  canal,  especially  the 
caecum,  of  poultry,  particularly  fowls  and  turkeys. 

Sarcina  urinae,  Welcker. — Very  small  cocci, 
i  '2  p,  in  diam.,  united  in  families  of  8  to  64. 
Observed  in  the  bladder. 

Sarcina  litoralis,  Oersted. — Cocci  1*2 — 2  ju,  in 
diam.,  bound  together  in  4  to  8  families,  which,  in 
their  turn,  may  unite  and  include  as  many  as  64 
tetrads.  Plasma  colourless  ;  in  each  cell  i — 4 
sulphur  granules.  Discovered  in  sea-water  con- 
taining putrefying  matter. 

Sarcina  Reitenbachii,  Caspary. — Cocci  about 
i '5  to  2*5  p,  in  diam.,  at  the  time  of  division 
lengthened  to  4  p.  Mostly  united  together  from 
4  to  8  in  number ;  occasionally  16  or  more.  Colour- 
less cell-wall,  lined  with  rose-red  layer  of  plasma. 
Found  on  rotting  water  plants. 

Sarcina  hyalina,  Kiitzing. — Cocci  round,  2*5 
p,  in  diam.,  almost  colourless.  United  in  families 
of  4  to  24  cells,  reaching  15  /iin  diam.  In  marshes. 

Sarcina  alba. — Small  cocci.  They  form  small 
white  colonies  on  nutrient  gelatine.  In  test-tube 
cultivations  they  grow  slightly  along  the  needle 

*  Zopf,  Die  Spaltpilze.     1885. 


SYSTEMATIC    AND    DESCRIPTIVE. 


213 


track,  but  are  heaped  up  on  the  surface  without 
liquefying  the  gelatine.     They  are  present  in  the 


air. 


In  man 


Genus  IV. — Micrococcus. 
SPECIES. 


ASSOCIATED  WITH  DISEASE  : — 

f  Micrococcus  in  scarlatina 
Micrococcus  in  measles 
Micrococcus  in  whooping  cough    . 
Micrococcus  in  haemophilia  neona- 

torum 

Micrococcus  in  typhus  . 
Micrococcus      in      acute      yellow 

atrophy 

Micrococcus  in  dental  caries . 
Micrococcus  in  gangrene 
Micrococcus  pyogenes  tenuis 
Micrococcus  in  rabies    . 
x  Micrococcus     of    septicaemia     in 

[      rabbits 

f  Micrococcus  of  pyaemia  in  rabbits 
In  animals  (  Micrococcus  of  progressive  suppu- 
ration in  rabbits 
Micrococcus  parvus  ovatus    . 
^Micrococcus  of  pyaemia  in  mice     . 
In  plants    .     Micrococcus  amylivorus 

UNASSOCIATED  WITH  DISEASE  : — 


Micrococcus  cyaneus 
Micrococcus  aurantiacus 
Micrococcus  chlorinus  . 
Micrococcus  violaceus  . 
Micrococcus  luteus 
Micrococcus  rosaceus    . 
Micrococcus  haematodes 
Micrococcus  candidus    . 
Micrococcus  candicans  . 
Micrococcus  foetidus 
Micrococcus  crepusculum 
Micrococcus  cinnabareus 
Micrococcus  flavus  liquefaciens 
Micrococcus  flavus  tardigradus 
Micrococcus  versicolor 
Micrococcus  viticulosus 
Micrococcus  lacteus  faviformis 
Micrococcus  fulvus 


Possibly  only  saprophytic. 


Pathogenic  (?). 


Pathogenic. 


Pathogenic  (?). 


Chromogenic  saprophytes. 


/  Simple  saprophytes. 


Micrococcus  in  scarlatina. — Cocci  have  been 
described  in  cases  of  scarlet  fever  as  being  present 


214  BACTERIOLOGY. 

in  the  blood,*  in  the  scales  of  the  desquamating 
epidermis,!  and  in  the  discharges  and  ulcerated 
tissue  of  the  throat. 

Micrococcus  in  measles. — Round  cocci  and 
diplococci  have  been  observed  in  the  catarrhal 
exudations,  in  the  papules  and  in  the  capillary 
vessels  of  the  skin,  and  in  the  blood  of  patients 
attacked  with  measles. J 

Micrococcus  in  whooping  cough. — Elliptical 
cocci  are  said  to  be  constantly  present  in  the  expec- 
toration of  persons  suffering  from  whooping  cough. § 

Micrococcus  in  haemophilia  neonatorum, 
Klebs. — A  coccus,  which  has  been  named  Monas 
hamorrhagicum ,  is  stated  to  be  characteristic  of 
this  disease. 

Micrococcus  in  typhus.  —  Actively  motile 
dumb-bell  cocci  have  been  described  in  the  blood, 
and  plugs  of  cocci  in  the  lymphatics  of  the  heart, 
in  cases  of  typhus  fever.  || 

Micrococcus  in    acute   yellow    atrophy.— 
Cocci    have  been    observed   in  the  vessels   of  the 
liver  in  this  disease.^ 

Micrococcus  in  gangrene.— Oval  and  round 
cocci  are  found,  which  form  zoogloea  in  the  depth 
of  gangrenous  tissues.  From  gangrene  of  the  lung 

*  Coze  and  Feltz,  Malad.  Infect.    1872. 
t  Pohl-Pincus,  Centralblatt  f.  d.  Med.  Wiss.     1883. 
j  Keating,  Phil.  Med.  Times.    1882.    Cornil  and  Babes,  Les  Bac- 
teries.    1 885 . 

§  Burger,  Berl.  Klin.  Woch.  1883. 
||  Molt,  Brit.  Med.  Journal.  1883. 
IT  Eppinger,  Prager  Vierteljahrsschrift.  1875. 


SYSTEMATIC    AND    DESCRIPTIVE.  215 

cocci  have  been  isolated,  which  form  greyish-white 
colonies  in  plate-cultivations  of  nutrient  gelatine. 
In  a  test-tube  of  nutrient  gelatine  a  growth  results 
chiefly  on  the  surface ;  the  cultivations  yield  a 
penetrating  odour. 

Micrococcus  pyogenes  tenuis,  Rosenbach. — 
Cocci,  which,  cultivated  on  agar-agar,  form  an 
opaque  streak  in  the  track  of  the  needle  with 
a  transparent  glassy  growth  extending  from  it. 
Occasionally  found  in  the  pus  of  closed  abscesses. 

Micrococcus  in  rabies.  —  Cocci  have  been 
described  in  connection  with  hydrophobia.  The 
cocci  were  observed  in  sections  of  the  spinal  cord 
of  rabid  dogs.  The  descriptions  given  by  different 
observers*  vary  considerably,  and  it  is  not  yet 
ascertained  whether  any  particular  coccus  is  con- 
stantly associated  with  the  disease.  Nor  have 
the  organisms  observed  in  stained  preparations 
been  cultivated  apart  from  the  diseased  animal. 
By  many,  however,  hydrophobia  is  believed  to  be 
due  to  the  presence  of  a  micro-organism,  and  re- 
searches which  are  being  carried  on  in  connection 
with  attenuation  of  the  virus  still  continue  to 
excite  the  keenest  interest  f  (p.  168). 

Micrococcus     of     septicaemia    in    rabbits, 

*  Hermann  Fol,  Academic  des  Sciences.  1885.  Babes,  Les 
Bacteries.  1886.  Dowdeswell,  Journ.  Royal  Microscop.  Soc. 
1886. 

t  Pasteur,  Comptes  Rendus.  1882.  Congres  de  Copenhague, 
1884.  Academic  des  Sciences.  1885,  1886.  Vignal,  "  Report  on  M. 
Pasteur's  Researches  on  Rabies  and  the  Treatment  of  Hydrophobia 
by  Preventive  Inoculation,"  Brit.  Med.  Journal.  1886. 


2l6  BACTERIOLOGY. 

Koch.* — Ellipsoidal  cocci  *8 — i  p  in  largest  diam. 
The  disease  was  produced  by  the  injection  of  putrid 
meat  infusion.  After  death  slight  cedema  was 
noted  at  the  site  of  injection,  slight  extravasation  of 
blood,  and  great  enlargement  of  the  spleen.  No 
emboli  or  peritonitis  resulted.  Masses  of  cocci 
were  found  in  the  capillaries  of  different  organs, 
especially  in  the  glomeruli  of  the  kidneys.  Rabbits 
and  mice  inoculated  with  blood  from  the  heart 
proved  susceptible  to  the  disease. 

Micrococcus  of  pyaemia  in  rabbits, 
Koch.f — Round  cocci  and  diplococci  "25  p.  in 
diam.  The  disease  was  produced  by  the  subcu- 
taneous injection  in  a  rabbit  of  distilled  water,  in 
which  the  skin  of  a  mouse  had  been  macerated. 
At  the  autopsy  there  were  found  great  infiltration 
around  the  site  of  injection,  peritonitis,  and  accu- 
mulations in  the  liver  and  lungs ;  in  short,  the 
appearances  of  pyaemia.  In  the  capillaries  of  the 
organs  examined,  masses  of  cocci  were  observed 
enclosing  blood  corpuscles  (Fig.  74).  Fresh  in- 
oculations in  rabbits  with  exudation-fluid,  or  blood 
from  the  heart,  reproduced  the  same  disease. 

Micrococcus  of  progressive  suppuration  in 
rabbits,  Koch.J — Cocci  only  about  -15  //,  in  diam., 
principally  in  thick  zooglcea.  The  disease  was 
induced  by  the  injection  into  rabbits  of  decom- 
posing blood.  At  the  place  of  injection  a  spread- 

*  Koch,  Wundinfect.  Krankheit.    1878. 
t  Ibid. 
\  Ibid. 


SYSTEMATIC    AND    DESCRIPTIVE.  217 

ing  abscess  formed,  which  was  fatal  to  the  animal  in 
about  twelve  days.  No  bacteria  were  observed  in 
the  blood,  but  in  the  walls  of  the  abscess  thick 
masses  of  cocci  were  found.  The  pus  is  infectious, 
causing-  the  same  disease  in  healthy  rabbits. 

Micrococcus  parvus  ovatus  (Bacillus  parvus 
ovatus,  Loffler). — Small  ovoid  cocci,  similar  to  the 
coccus  of  rabbit  septicaemia.  Cultivated  on  gelatine 
they  develop  readily  a  greyish-white  growth  at  the 


FIG.  74. — MICROCOCCUS  OF  PYAEMIA  IN  RABBITS  ;  VESSEL  FROM  THE 
CORTEX  OF  THE  KIDNEY,  x  700.  (a)  Nuclei  of  the  vascular  wall ;  (c) 
Masses  of  micrococci  adherent  to  the  wall  and  enclosing  blood  corpuscles. 

entrance  of  the  inoculating  needle.  They  are 
pathogenic  in  mice  and  rabbits.  They  proved  also 
fatal  in  a  pig  after  two  days,  producing  oedema  of 
the  skin,  inflammation  of  the  mucous  membrane  of 
the  stomach,  but  no  effect  on  the  general  intestinal 
tract  or  mesentery.  They  were  isolated  from  a  pig 
suffering  from  a.  fatal  disease  simulating  swine- 
erysipelas. 

Micrococcus   of  pyaemia  in   mice,  Klein. — 
Certain    cocci    which    were  present  in   pork  broth 


2  1 8  BACTERIOLOGY. 

proved  fatal  to  mice  in  about  a  week,  producing 
purulent  inflammation  and  abscess  in  the  lungs. 
Fresh  inoculations  in  mice  again  produced  a  fatal 
result  with  pysemic  symptoms. 

Micrococcus  amylivorus,  Burrill. — Oval  cells, 
i — i -4  n  long,  *7  */x  broad,  singly,  in  pairs,  and 
rarely  in  fours,  never  in  chains,  are  found  em- 
bedded in  an  abundant  mucilage  which  is  very 
soluble  in  water.  They  have  been  described  as 
producing  the  so-called  "fire  blight"  of  the  pear 
tree  and  other  plants. 

Micrococcus  cyaneus,  Cohn  (Baderidium 
cyaneum,  Schroter). — Elliptical  cells,  growing  upon 
cooked  potato,  and  producing  a  blue  colour.  In 
nutrient  solutions  they  form  zooglcea,  at  first 
colourless,  then  bluish-green,  and  finally  intense 
blue. 

Micrococcus  aurantiacus,  Schroter. — Cocci, 
oval,  i '5  p  in  diam.,  singly  or  in  pairs,  or  in 
zooglcea.  They  occur  as  orange-yellow  spots  which 
coalesce  into  patches.  A  golden-yellow  pellicle 
develops  when  they  are  cultivated  in  nutrient 
liquids.  The  colouring  matter  is  soluble  in  water. 
They  were  observed  on  boiled  potatoes  and  white 
of  egg. 

Micrococcus  chlorinus,  Cohn. — Cocci  occur 
in  the  form  of  a  finely  granular  zooglcea,  causing  a 
yellowish  green  or  sap-green  layer  on  boiled  eggs 
and  nourishing  solutions.  The  colouring  matter 
is  soluble  in  water  and  is  decolorised  by  acids. 


SYSTEMATIC    AND    DESCRIPTIVE.  2ig 

Micrococcus  violaceus,  Schroter.  —  Cocci 
or  elliptical  cells,  described  as  uniting  into  violet 
blue  gelatinous  spots,  which  again  unite  to  form 
larger  patches.  They  were  observed  on  boiled 
potatoes  exposed  to  the  air. 

Micrococcus  luteus,  Schroter. — Cocci  similar 
in  size  to  the  above,  elliptical,  with  highly  refrac- 
tive cell  contents.  They  form  yellow  drops  of 
i — 3  mm.  diam.  on  boiled  potato,  and  a  thick, 
wrinkled,  yellow  skin  on  nutrient  liquids.  The 
colouring  matter  is  insoluble  in  water,  and  un- 
changed by  sulphuric  acid  or  alkalies. 

Micrococcus  rosaceus. — Cocci  forming  pink 
colonies,  and  a  rose-coloured  growth  on  the  surface 
of  nutrient  agar-agar.  Observed  contaminating 
an  old  cultivation  (Plate  XXIV.,  Fig.  2). 

Micrococcus  haematodes,  Zopf.  —  Cocci, 
which,  cultivated  on  boiled  white  of  egg  in  a 
damp  chamber  in  the  incubator,  form  a  red  layer. 
The  reaction  of  the  colouring  matter  is  similar  to 
that  produced  by  Bacterium prodigiosum.  They  have 
been  observed  in  human  sweat,  especially  from  the 
axilla,  colouring  the  surrounding  parts  and  the 
linen  an  intense  brick  or  blood-red  colour.* 

Micrococcus  candidus,  Cohn. — Cocci  forming 
snow-white  points  and  spots,  upon  slices  of  cooked 
potato.  Possibly  identical  with  the  following  : — 

Micrococcus  candicans,Flugge. — Cocci  which 
collect  in  masses.  In  plate-cultivations  they  form 

*  Babes,  "  Vom  Rothen  Schvveiss,"  Biol.  CcntrabL,  Bd.  2.     1882. 


220  BACTERIOLOGY. 

in  two  or  three  days  milk-white  colonies.  Culti- 
vated in  test-tubes  they  form  a  white  nail-shaped 
cultivation.  They  were  isolated  from  contaminated 
plate-cultivations. 

Micrococcus  fcetidus,  Rosenbach.  —  Small 
oval  cocci.  Cultivated  in  agar-agar  they  develop 
gas  bubbles  and  a  foetid  odour.  Isolated  from 
carious  teeth.  Possibly  closely  allied  to,  if  not 
identical  with,  the  following. 

Micrococcus  crepusculum,  Cohn  (Monas 
crepusculum,  Ehrenberg.  Mikrokokken  in  faulenden 
Substraten,  Fliigge).  Round  or  short  oval  cells, 
scarcely  2  p,  in  diam.  ;  singly  or  in  zooglcea. 
They  occur  in  various  infusions  and  putrefying 
fluids  in  company  with  Bacterium  termo. 

Micrococcus  cinnabareus,  Fliigge.  —  Large 
cocci  occurring  in  twos,  threes,  and  fours.  In 
plate-cultivations  they  grow  very  slowly,  forming 
punctiform  colonies,  in  colour  bright  red  at  first, 
and  afterwards  reddish-brown.  In  test-tube  culti- 
vations they  form  on  the  surface  of  the  gelatine 
a  heaped-up  red-coloured  growth.  Found  con- 
taminating old  cultivations. 

Micrococcus  flavus  liquefaciens,  Fliigge. — 
Cocci,  diplococci,  and  zoogloea.  On  plate-cultiva- 
tions they  form  yellowish  colonies,  and  in  test-tubes 
yellowish  beads,  which  become  confluent  and 
rapidly  liquefy  the  gelatine. 

Micrococcus  flavus  tardigradus,  Fliigge. — 
Cocci  forming  chrome-yellow  colonies.  Cultivated 


SYSTEMATIC    AND    DESCRIPTIVE.  221 

in  test-tubes  they  form  yellowish  beads  in  the 
needle  track,  which  remain  isolated,  and  do  not 
liquefy  the  gelatine.  Isolated  from  contaminated 
cultures. 

Micrococcus  versicolor,  Fliigge.  — Small 
cocci,  which  form  iridescent  colonies.  In  test-tubes 
they  grow  in  the  form  of  yellowish  beads,  and 
develop  an  iridescent  layer  on  the  surface. 

Micrococcus  viticulosus,  Fliigge.  —  Oval 
cocci,  1*2  /i  in  length,  and  i  ^  in  width.  In  plate- 
cultivations  the  colonies  differ  when  embedded  in 
the  nutrient  medium  and  when  growing  on  the 
surface.  The  characteristic  appearance  consists  of 
a  delicate  network,  which  is  visible  also  in  test- 
tube  cultivations  in  the  track  of  the  needle.  On 
the  surface  of  the  medium  they  form  a  viscous 
layer.  They  were  isolated  from  contaminated 
cultivations. 

Micrococcus  lacteus  faviformis,  Bumm  and 
Bockart. — Cocci  1*25  //,  in  diam.  Cultivated  in 
gelatine  they  form  milk-white  confluent  colonies, 
and  preparations  made  from  the  cultivations  have 
a  characteristic  honeycomb  appearance.  Isolated 
from  vaginal  secretions,  and  from  sputum. 

Micrococcus  fulvus,  Cohn.  —  Cocci  round 
i  "5  fj,  in  diam.,  frequently  in  pairs.  They  form 
rusty-red  conical  drops  of  a  firm  consistency,  and 
about  *5  mm.  diam.,  on  horse  dung. 


222 


BACTERIOLOGY. 


Genus   V. — Ascococcus. 


SPECIES. 


UNASSOCIATED  WITH  DISEASE  : — 
Ascococcus  Billrothii 


Zymogenic  Saprophyte. 


Ascococcus  Billrothii. — Small  globular  cocci, 
united  into  characteristic  colonies.  They  form  on 
the  surface  of  nourishing  fluids  a  cream-like  skin, 
divisible  into  an  enormous  number  of  globular  or 
oval  families.  Each  family  is  surrounded  by  a 
thick  capsule  of  cartilaginous  consistency.  In  a 


m 

'''«•!...••* 

FIG.  75. — Ascococcus  BILLROTHII  [after  Cohn]. 

solution  containing  acid  tartrate  of  ammonia  the 
fungi  generate  butyric  acid,  and  change  the  origi- 
nally acid  fluid  into  an  alkaline  one.  They  were 
first  observed  on  putrid  broth,  and  later  on  or- 
dinary nourishing  solutions ;  they  also  readily 
develop  upon  damp  slices  of  boiled  roots,  carrots, 
beetroots,  etc. 


SYSTEMATIC    AND    DESCRIPTIVE.  22J 

METHODS    OF    STAINING    COCCI. 

Cocci  stain  well  with  watery  solutions  of  gentian-violet, 
methyl-violet,  fuchsine,  methylene  blue,  and  bismarck 
brown.  For  examining  cocci  in  liquids  such  as  pus  or 
blood,  or  in  cultivations  in  solid  media,  a  little  of  the 
material  should  be  spread  out  on  a  cover-glass  (page  48), 
and  stained  with  a  drop  or  two  of  a  watery  solution  of 
fuchsine  or  methyl-violet.  The  former  is  especially  recom- 
mended for  staining  Merismopedia  gonorrhoea. 

For  a  zooglcea,  or  pellicle  of  micrococci,  Klein  recom- 
mends transference  bodily  to  a  watch-glass  containing  the 
dye,  leaving  it  there  till  deeply  tinted,  then  taking  it  out 
with  a  needle,  washing  in  water,  and  then  in  alcohol  till 
excess  of  colour  is  removed.  It  must  then  be  transferred 
to  a  glass-slide,  spread  well  out,  and  a  drop  of  clove-oil 
placed  on  it ;  after  a  minute  or  two  the  clove-oil  is  drained 
off,  a  drop  of  Canada  balsam  added,  and  covered  with  a 
cover-glass.* 

Cocci  in  the  tissues  may  be  stained  by  immersing  the 
sections  in  an  aqueous  solution  of  gentian-violet,  or 
in  aniline-gentian-violet  solution,  then  rinsing  in  water, 
decolorising  in  alcohol,  treating  with  clove-oil,  and  pre- 
serving in  balsam  (p.  58)  ;  or,  after  washing  with  alcohol, 
they  may  be  rinsed  with  water,  and  stained  for  half  an 
hour  with  Weigert's  picrocarmine.  From  this  they  are 
again  removed  to  water,  then  to  alcohol,  clove-oil,  and 
Canada  balsam. 

The  method  of  Gram  is  much  more  satisfactory  (p.  59, 
Plate  XII.,  Fig.  2).  Sections  should  be  examined  with 
and  without  a  contrast  stain.  The  after-stain  most  com- 
monly employed  is  eosin.  The  sections  after  the  process 
of  decolorisation  should  be  placed  in  a  weak  alcoholic 
solution  of  eosin  (two  or  three  drops  of  a  concentrated 
alcoholic  solution  added  to  a  watch-glassful  of  alcohol), 
till  stained  a  delicate  pink.  They  are  then  rinsed  in 
*  Klein,  Micro-organisms  and  Disease.  1885. 


224  BACTERIOLOGY. 

fresh  alcohol,  treated  with  clove-oil,  and  preserved  in 
Canada  balsam. 

Sections  containing  cocci  of  osteomyelitis  may  be  after- 
stained  w\\h.  weak  solution  ofvesuvin.  Safranine  and  picro- 
lithium-carmine  may  also  be  used  as  contrast  stains  (p.  61). 

Nuclear  stains,  such  as  carmine,  hsematoxylin,  may  also 
be  employed.  Sections  may  be  left  one  minute  in 
Grenacher's  solution,  then  washed  out  in  weakly  acidulated 
alcohol  (2 — 1000)  ;  and  finally  treated  in  the  usual  way, 
with  alcohol,  oil  of  cloves,  and  balsam. 

Sections  containing  micrococcus  tetragonus  are  best 
stained  with  Gram's  method  and  eosin  (Plate  XII.,  Fig.  l), 
but  they  may  also  be  treated  by  the  method  of  Fried- 
lander,  to  demonstrate  their  capsules  (p.  227). 

To  stain  the  cocci  of  rabbit-septic&mia  in  the  tissues, 
place  the  sections  twenty-four  hours  in  Loffler's  solution, 
wash  in  water  faintly  acidulated  with  acetic  acid,  then  treat 
with  alcohol,  oil  of  cloves,  and  balsam. 

GROUP  II. — BACTERIACE^:. 

Genus  I.  Bacterium. — Cocci  and  rods,  or  only  rods, 
which  are  joined  together  to  form  threads.  Spore- 
formation  absent  or  unknown. 

Genus  II.  Spirillum. — Threads  screw-form,  made  up 
of  rods  (long  or  short)  only,  or  of  rods  and  cocci. 
Spore-formation  absent  or  unknown. 

Genus  III.  Leuconostoc. — Cocci  and  rods.  Spore- 
formation  present  in  cocci. 

Genus  IV.  Bacillus. — Cocci  and  rods,  or  rods  only, 
forming  straight  or  twisted  threads.  Spore- 
formation  present  either  in  rods  or  cocci. 

Genus  V.  Vibrio. —Threads  screw-form  in  long  or 
short  links.  Spore-formation  present. 

Genus  VI.  Clostridium. — Same  as  bacillus,  but  spore- 
formation  takes  place  in  characteristically  en- 
larged rods. 


SYSTEMATIC    AND    DESCRIPTIVE 


225 


Genus  I. — Bacterium. 


SPECIES. 
ASSOCIATED  WITH  DISEASE  : — 

Bacterium  pneumonia  crouposse 


In  man 


In  animals  ( 


Bacterium  pseudo-pneumonicum  . 

Bacterium  Neapolitanum 
Bacterium  in  rhinoscleroma . 
Bacterium  in  diphtheria 

Bacterium  saprogenes   • 

Bacterium  decalvans 
^Bacterium  in  diphtheria  of  calves 
Bacterium  of  diphtheria  of  pigeons 
Bacterium  cholerae  gallinarum 
Bacterium  septicum  agrigenum 
Bacterium  of  septicoemia  in  rabbits 
Bacterium  of  Davaine's  septicaemia 
Bacterium  septicum  sputigenum    . 


Bacterium  crassum  sputigenum 

Bacterium  pneumonicum  agile 

Bacterium  oxytocum  perniciosum . 

Bacterium  cavicida 

Bacterium  coli  commune 

Bacterium  lactis  aerogenes    . 

Panhistophyton  ovatum 
In  plants  .      Bacterium  hyacinth! 
UNASSOCIATED  WITH  DISEASE  : — 

Bacterium  synxanthum 

Bacterium  indicum 

Bacterium  rubrum 

Bacterium  prodigiosum 

Bacterium  luteum 

Bacterium  violaceum    . 

Bacterium  brunneum    . 

Bacterium  fluorescens  putidum 

Bacterium  fluorescens  liquefaciens 

Bacterium  ureae     .... 

Bacterium  aceti     .... 

Bacterium  Pasteurianum 

Bacterium  liodermos 

Bacterium  multipediculum    . 

Bacterium  ramosum  liquefaciens    . 

Bacterium  Zopfii  . 

Bacterium  merismopedioides 

Bacterium  Pfliigeri 

Bacterium  photometricum     . 

Bacterium  litoreum 

Bacterium  fusiforme 

Bacterium  navicula 

Proteus  vulgaris    .... 

Proteus  mirabilis  . 

Proteus  Zenkeri    .... 

Bacterium  termo  .... 

Bacterium  lineola 


Pathogenic  (?) ;  possibly  only 
saprophytic  in  man,  patho- 
genic in  animals. 

Saprophytic  in  man,  patho- 
genic in  animals. 

»  >»  »» 

Pathogenic  in  man  (?). 

Saprophytic  in  man  (?), 
pathogenic  in  animals. 

Saprophytic  in  man,  patho- 
genic in  animals. 

Saprophytic. 

Pathogenic  (?). 

Pathogenic. 


Pathogenic  (?). 


Chromogenic  saprophytes. 


Zymogenic  saprophytes. 


Simple  saprophytes. 


226  BACTERIOLOGY. 

Bacterium  pneumoniae  crouposae  (Pneumo* 
coccus,  Friedlander). — Cocci  ellipsoidal  and  round, 
singly,  or  in  pairs  (diplococci),  rods  and  thread 
forms.  The  cell- membrane  thickens,  and  develops 
into  a  gelatinous  capsule,  which  is  round  if  the 
coccus  is  single,  and  ellipsoidal  if  the  cocci  occur  in 
pairs  or  in  rod-forms  (Fig.  76,  Plate  I.,  Fig.  5). 
Cultivated  in  a  test-tube  of  nutrient  gelatine  they 
grow  along  the  needle  track  in  the  form  of  a  round- 
headed  nail  (Plate  IV.,  Fig.  2),  without  liquefaction 


FIG.  76.— BACTERIUM  PNEUMONIA  CROUPOSAE,  FROM  PLEURAL  CAVITY 
OF  A  MOUSE,  x  1500.  A,  B.  Thread-forms.  C,  D,  E.  Short  rod-forms. 
G.  Diplococci.  H.  Cocci.  I.  Streptococci.  [After  Zopf.] 

of  the  gelatine.  The  cocci  when  artificially  culti- 
vated have  no  capsule,  but  it  again  appears  after 
their  injection  into  animals.  The  cocci  can  also 
be  cultivated  on  blood  serum  and  on  boiled  potatoes. 
They  occur  in  pneumonic  exudation.*  Inoculation 
of  dogs  with  a  cultivation  of  the  cocci  occasionally 
gave  positive  results ;  but  in  rabbits  no  results 

*  Friedlander,  Fortschr.  d.  Med.    1883. 


SYSTEMATIC    AND    DESCRIPTIVE.  227 

followed.  Guinea-pigs  proved  to  be  susceptible 
in  some  cases,  but  thirty-two  mice,  after  injection 
of  a  cultivation  diffused  in  sterilised  water  into  the 
lungs,  died  without  exception.  The  lungs  were 
red  and  solid,  and  contained  the  cocci,  which  were 
also  present  in  the  blood,  and  in  enormous  numbers 
in  the  pleural  exudation.  Inhalation  experiments 
by  spraying  the  cocci  diffused  in  water  into  mouse 
cages  succeeded  in  producing  pneumonia  and 
pleurisy  in  three  out  of  ten  mice.  The  nail-shaped 
cultivation  is  not  always  produced,  nor  are  these 
conclusions  accepted  by  all  investigators.* 

METHODS    OF    STAINING    THE    BACTERIA    OF 
PNEUMONIA. 

(Pneumonic- Coccen,  Friedlander.) 

Cover-glass-preparations  (p.  48)  of  pneumonic  sputum 
or  exudation  may  be  treated  as  follows : — 

(a)  Stain  by  the  method  of  Gram,  and  after-stain  with 
eosin  (p.  59). 

(U)  Treat  with  acetic  acid,  then  stain  with  gentian-violet 
or  bismarck-brown.  Examine  in  distilled  water,  or  dry  and 
preserve  in  Canada  balsam. 

(c)  Float  them  on  weak  solutions  of  the  aniline  dyes 
twenty-four  hours ;  differentiation  between  coccus  and 
capsule  is  thus  obtained. 

(//)  Stain  with  osmic  acid;  the  contour  of  the  capsules 
is  brought  out. 

Sections  of  pneumonic  lung  should  be  stained  by 

(a)  Method  of  Gram. 

(b)  Method  of  Friedlander.     This  method  is  employed 
to  demonstrate  the  capsules  in  tissue  sections.     It  consists 

*  Klein,  Micro-organisms-  and  Disease.     1885. 


228  BACTERIOLOGY. 

in  placing  the  sections  twenty-four  hours  in  the  following 
solution  : — 

Fuchsine         .         .      '.»'-..         .         i 
Distilled  water       .         .         *        .100 

Alcohol 5 

Glacial  acetic  acid          .         .  2 

They  are  then  rinsed  with  alcohol,  transferred  for  a  couple 
of  minutes  to  a  2  per  cent,  solution  of  acetic  acid,  and  in 
the  usual  way  treated  with  alcohol  and  oil  of  cloves,  and 
preserved  in  Canada  balsam. 

Bacterium  pseudo-pneumonicum  (Bacillus 
pseudo-pneumonicus,  Passet).  Cocci  round,  oval,  and 
occasionally  elongated,  similar  to  the  bacterium 
of  pneumonia.  The  oval  forms  are  "87  ft  in  width, 
and  i*i6ft  in  length.  The  colonies  on  plates  appear 
in  twenty- four  hours  as  white  dots  ;  in  test-tubes 
the  growth  develops  as  a  greyish- white  layer. 
If  injected  into  the  pleura  they  set  up  pleuritis, 
and  into  the  abdomen  peritonitis,  in  mice,  rats,  and 
guinea-pigs.  Subcutaneous  inoculation  produces 
septicaemia  in  mice,  and  abscesses  in  rats,  guinea- 
pigs,  and  rabbits.  Inhalation  experiments  gave 
no  results.  They  were  isolated  from  pus. 

Bacterium  Neapolitanum  (Bacillus  Neapoli- 
tanus,  Emmerich).  Short  rods  with  rounded  ends. 
In  width  *9  p,  (Fig.  77).  They  form  circular  colonies, 
which  later  become  irregular,  granular,  strongly 
refractive,  and  of  a  yellowish-brown  colour.  By 
introducing  a  large  quantity  into  small  animals 
changes  were  produced  in  the  intestines  with  an 


SYSTEMATIC    AND    DESCRIPTIVE.  2 29 

analogy  to  the  post-mortem  appearances  of  cholera. 
They  are  probably  identical  with  bacteria  found  in 
healthy  faeces.  They  were  isolated  from  some  cases 
of  cholera  at  Naples. 


*  *!o 

* i  «*\ 

a 

FIG.  77. — BACTERIUM  NEAPOLITANUM,  x  700.  (a)  From  intestinal  con- 
tents in  a  case  of  cholera  ;  (£)  From  peritoneal  fluid  of  an  inoculated 
guinea-pig  [after  Emmerich]. 

Bacterium  of  Rhinoscleroma  (Bacillus  of 
Rhinoscleroma,  Cornil  and  Alvarez  *).  Cocci  and 
short  rods,  1*5 — 3  //,  in  length,  -5 — '8  /x  thick. 
Deeply  coloured  points  or  granules  may  occur  in 
the  course  of  the  rods  when  stained,  but  it  is  very 
doubtful  whether  these  can  be  considered  as  spores. 


FIG.  78.— BACTERIA  OF  RHINOSCLEROMA,  x  1400.     Encapsuled  cocci,  diplo- 
cocci,  and  short  and  long  rod-forms  [after  Cornil]. 

The  bacteria  are  encapsuled,  the  capsule  being  round 
when  enclosing  a  coccus,  and  ovoid  when  enclosing 
a  rod  (Fig.  78).  The  capsule  is  composed  of  a  tough 
resisting  substance;  two  or  more  capsules  may 
unite  by  fusion,  enclosing  two  or  three,  or  a  great 

*  Cornil  and  Babes,  Les  Bacteries.     1885. 


23O  BACTERIOLOGY. 

number  of  rods.  The  bacilli  were  observed  in 
sections  of  a  tumour,  rhinoscleroma,  which  develops 
on  the  lip  and  on  the  nasal  and  pharyngo-laryngeal 
regions. 

METHOD    OF    STAINING    THE    BACILLUS    OF    RHINO- 
SCLEROMA. 

Method  of  Cornil  and  Alvarez : — 

Sections  arc  immersed  in  a  solution  of  methyl-violet  (B) 
for  twenty-four  to  forty-eight  hours,  with  or  without  the 
addition  of  aniline-water;  are  then  decolorised  after  treat- 
ment with  the  solution  of  iodine  in  iodide  of  potassium. 
If  the  sections  are  left  to  decolorise  in  alcohol  for  forty- 
eight  hours  the  capsule  is  rendered  visible. 

Bacterium  in  diphtheria  of  man  (Bacillus 
of  diphtheria,  Lo  frier). — Rods  about  the  same  length 
as  the  tubercle  bacillus,  but  about  twice  as  thick; 
the  longer  ones  consist  of  single  individuals 
linked  together.  Spores  not  observed.  They 
were  cultivated  in  a  mixture  consisting  of  three 
parts  of  calf's  or  lamb's  blood  serum,  to  which 
was  added  one  part  of  neutralised  veal  broth, 
containing  i  per  cent,  peptone,  i  per  cent,  grape 
sugar,  \\  per  cent,  common  salt.  Cultivated  in 
5  per  cent,  gelatine  at  20 — 22°  C.,  the  rods 
developed  into  irregular  involution-forms.  In- 
oculation gave  doubtful  results.  The  bacillus  was 
isolated  from  diphtheritic  membrane.*  They  were 
particularly  noticeable  in  those  typical  cases 

*  Loffler,  Mittheil.  a.d.  K.  Gesundheitsamte  (Microfiarasites  in 
Disease,  New  Syd.  Society). 


SYSTEMATIC    AND    DESCRIPTIVE.  23  I 

characterised  by  a  thick  false  membrane,  extending 
over  the  fauces,  larynx,  and  trachea.  They  occupied 
the  deeper  layers  below  the  masses  of  bacteria 
which  are  found  on  the  surface,  such  as  the 
Streptococci  already  described  (p.  202). 

METHOD  OF  STAINING  THE  BACTERIA  IN   DIPHTHERIA. 

Lofflers  Method:— 

Sections  are  placed  in  Loffler's  solution  for  a  few  minutes, 
and  excess  of  stain  removed  by  \  per  cent,  solution  of  acetic 
acid.  They  are  then  treated  with  alcohol  and  cedar-oil,  and 
mounted  in  Canada  balsam. 

Bacterium  saprogenes  (Bacillus  saprogenes 
No.  3,  Rosenbach). — Rods  isolated  from  the  putrid 
marrow  of  a  case  of  compound  fracture.  Cultivated 
on  nutrient  agar-agar,  an  ash-grey,  almost  liquid 
culture  is  developed,  with  a  strong  characteristic 
odour  of  putrefaction.  Injected  into  the  knee  joint 
or  abdomen  of  a  rabbit,  an  opaque,  yellowish-green 
infiltration  resulted  (vide  Bacillus  saprogenes,  p.  3  14). 

Bacterium  decalvans,  Thin. — Cocci,  singly  or 
in  pairs,  1*6  JJL  in  length.  Observed  in  the  roots  of 
the  hair  in  cases  of  Alopecia  areata. 

Bacterium  in  diphtheria  of  calves  (Bacillus 
vitulorum,  Loffler). — Rods  about  five  or  six  times  as 
long  as  wide,  mostly  united  in  long  threads.  A 
piece  of  tissue  placed  on  blood  serum  developed  a 
white  layer  composed  of  the  bacilli.  Successive 
generations  were  not  obtainable.  Mice  inoculated 
directly  from  the  calf  died  of  a  characteristic  illness, 


232  BACTERIOLOGY. 

and  the  same  long  bacteria  were  again  found  in  the 
inoculated  animals  accompanying  widespread  infil- 
tration, starting  from  the  point  of  inoculation. 
Inoculation  of  guinea-pigs  and  rabbits  gave  doubt- 
ful results.  The  bacteria  were  found  in  the  deeper 
stratum  of  the  diphtheritic  patches. 

Bacterium  of  diphtheria  of  pigeons  (Bacillus 
co  lumbar um^  Loffler). — Short  rods  with  rounded 
ends,  mostly  in  irregular  masses.  In  plate-cultiva- 
tions on  nutrient  gelatine  they  formed  whitish 
patches  on  the  surface,  and  compact,  ball-like 
masses  when  embedded  in  the  gelatine.  They 
were  also  cultivated  ,on  blood  serum  and  potatoes. 
Subcutaneous  inoculations  in  pigeons  with  a  pure 
cultivation  produced  local  inflammation  and 
necrosis ;  inoculation  in  the  mucous  membrane  of 
the  mouth  gave  the  appearances  of  the  original 
disease.  Other  animals  were  only  locally  affected, 
except  mice,  in  which  characteristic  symptoms 
and  death  resulted.  They  were  isolated  from 
the  diphtheritic  exudations  in  pigeons,  and  in 
sections  were  found  in  the  vessels  of  the  lungs 
and  liver. 

Bacterium  cholerae  gallinarum  (Micrococcus 
cholera  gallinarum,  Zopf.  Bacterium  of  Fowl- cholera. 
Microbe  du  chottra  despoules). — Cocci  2 — 3  p.  in  diam., 
short  rods  staining  deeply  at  either  pole,  and  longer 
beaded  rods  (Figs.  79, 80).  In  the  tissues  they  appear 
mostly  as  rods  2  to  3  //,  in  length  and  5  p  in  diam., 
with  their  extremities  stained  more  deeply  than 


SYSTEMATIC    AND    DESCRIPTIVE.  233 

their  middle*  (vide  p.  135).  When  cultivated  by 
introducing  a  drop  of  the  infected  blood  into  sterile 
chicken- broth,  a  number  of  round  bodies,  undergoing 
rapid  movement  and  as  a  rule  united  as  diplococci, 
or  elongated  and  contracted  in  the  middle,  appear 
in  the  broth,  which  is  at  first  slightly  milky,  but  be- 


FIG.  79.  —  BACTERIUM  OF  CHICKEN  CHOLERA  ;   BLOOD  OF 
INOCULATED  HEN,    x  1200. 

comes  limpid,  and  the  microbes  at  the  same  time 
pass  into  a  finely  granular  state.  From  this,  how- 
ever, fresh  cultures  can  still  be  started.  Cultivated 
in  a  test-tube  of  nutrient  gelatine,  after  from  three 


FIG.  80.  —  BACTERIUM  OF  CHICKEN  CHOLERA,  FROM  MUSCLE  JUICE 
OF  INFECTED  HEN.  x  2500  [from  a  Photograph]. 

days  to  a  week  there  develops  along  the  needle 
track  a  fine,  almost  imperceptible,  greyish  thread 
without  liquefaction  of  the  gelatine  (Plate  III., 
Fig.  2).  The  growth  is  exceedingly  scanty,  even 
after  several  weeks. 

Fowls  suffering  from  the  disease  usually  die  very 

*  Cornil  and  Babes,  Les  Bacteries. 


234  BACTERIOLOGY. 

rapidly.  In  the  less  acute  cases  they  are  somnolent, 
weak  in  their  legs,  and  their  wings  trail.  They 
suffer  from  diarrhoea,  and  pass  into  a  state  of  sopor 
and  die.  The  micro-organisms  are  found  in  large 
numbers  in  the  blood  and  organs  after  death,  and 
in  the  intestinal  discharges. 

A  drop  of  the  broth  injected  into  the  connective 
tissue  in  the  region  of  the  pectoral  muscles  causes 
the  death  of  the  fowl  the  following  day,  with  charac- 
teristic pathological  changes.*  If  a  culture  be  kept 
for  some  time,  and  a  fowl  be  then  inoculated  with 
it,  instead  of  death  only  local  changes  are  produced, 
and  the  fowl  is  protected  against  the  action  of  a 
virulent  culture ;  thus  affording  an  example  of  so- 
called  mitigation  of  the  virus. ,f  The  microbe  is 
aerobic,  and  its  toxic  effect  has  been  supposed  to  be 
due  to  the  abstraction  of  oxygen  from  the  blood 
producing  asphyxia. 

Bacterium  septicum  agrigenum  (Bacillus 
septicus  agrigenus,  Nicolaier). — Cells  morphologi- 
cally similar  to  the  microbe  of  chicken  cholera. 
The  colonies  on  plate-cultivations  have  a  yellowish- 
brown  centre,  with  a  greyish-yellow  zone.  In  test- 
tube  cultivations  the  appearances  are  not  charac- 
teristic. They  are  pathogenic  in  mice  and  in 
rabbits.  The  organs  show  no  characteristic  post- 


*  Cornil,  "  Observ.  Hist,  sur  les  Lesions  des  Muscles  determinees 
par  Pinjection  du  Microbe  du  Cholera  des  Poules  "  (Archives  de 
Physiologic.  1882) ;  Cornil  and  Babes,  Les  Bacteries.  1885. 

t  Pasteur,  <(  Sur  le  Cholera  des  Poules,"  Compt.  Rendus*    1880. 


SYSTEMATIC    AND    DESCRIPTIVE.  235 

mortem  appearances,  but  the  bacteria  abound  in  the 
blood.     They  were  isolated  from  earth. 

Bacterium  of  septicaemia  in  rabbits  (Bacillus 
cuniculicida,  Koch). — Short  rods,  slightly  pointed  at 
both  ends ;  in  width  '6  JJL — *7  /x,  in  length  i  '4  JJL.  They 
stain  deeply  at  the  ends,  leaving  an  uncoloured  in- 
terval in  the  middle  (Fig.  81), — an  appearance  which 
must  be  distinguished  from  a  diplococcus  or  figure 
of  eight.  Two  or  more  bacteria  may  be  linked 
together  in  a  chain.  They  may  be  cultivated  in 
bouillon,  blood  serum,  and  nutrient  gelatine.  In 
plate-cultivations  of  the  latter,  they  produce  dot- 


FIG.  81. — BACTERIUM  OF  RABBIT  SEPTICAEMIA;  BLOofe  OF  SPARROW, 
X  700  [after  Koch]. 

like  colonies,  and  in  test-tubes  little  spherical 
masses  in  the  needle  track,  and  a  layer  on  the  free 
surface.  The  smallest  quantity  inoculated  subcu- 
taneously  or  in  the  cornea  of  a  rabbit  produces 
a  rise  of  temperature  and  laboured  breathing  after 
10 — 12  hours,  and  death  in  16 — 20  hours.  The 
spleen  and  lymphatic  glands  are  found  to  be  en- 
larged, and  the  lungs  congested,  but  no  extravasa- 
tions, and  no  peritonitis.  In  the  blood  the  charac- 
teristic rods  abound,  and  in  sections  they  are  found 
in  the  vessels  and  capillaries.  Mice  and  birds  are 
very  susceptible ;  guinea-pigs  and  white  rats  have 


236  BACTERIOLOGY. 

an  immunity.  The  disease  was  produced  by  inocu- 
lating rabbits  with  contaminated  water  (River 
Panke)  and  with  putrid  meat  infusion. 

Bacterium  of  Davaine's  septicaemia. — Rods 
similar  to  the  bacteria  described  by  Koch.  They 
were  also  found  in  the  blood  of  rabbits  suffering 
from  septicaemia,  which,  however,  differed  from 
Koch's  septicaemia  in  that  guinea-pigs  were  sus- 
ceptible, and  pigeons  immune. 

Bacterium  septicum  sputigenum  (Microbe  de 
saiive^  Pasteur.  Micrococcus  Pasteuri,  Sternberg.* 
Bacillus  septicus  sputi genus,  Frank  el). — Cocci  oval, 
singly,  in  pairs,  and  in  chains ;  often  lanceolate  or 
rod-shaped;  encapsuled.  They  grow  well  in  broth, 
and  on  agar-agar  at  30°  to  35°  C.  On  the  solid 
media  they  form  a  superficial,  nearly  transparent 
deposit  of  gelatinous  consistence.  They  are 
pathogenic  in  rabbits,  producing  typical  "  sputum 
septicaemia."  Fowls  and  dogs  have  an  immunity, 
and  guinea-pigs  are  less  susceptible  than  rabbits. 
Mice  die  within  forty-eight  hours  after  being  inocu- 
lated. The  blood  of  an  infected  rabbit  just  dead 
is  more  potent  than  a  liquid  culture  or  than  saliva 
containing  the  coccus.  An  animal  which  recovers 
after  an  injection  of  saliva  is  stated  to  be  protected 
from  the  potent  virus.  The  pathogenic  power  is 
modified  by  cultivation  at  a  temperature  between 
39*5°  and  4O'5°.  The  organism  has  been  supposed 

*  Sternberg,  Stud.  BioL  Lab.,  John  Hopkins,  Univ.  n,  No.  2, 
1882.     Journal  Royal  Microscop.  Society.    1886. 


SYSTEMATIC    AND    DESCRIPTIVE.  237 

to  be  intimately  associated  with  croupous  pneu- 
monia, but  any  exact  relation  cannot  be  considered 
as  established.  The  organism  differs  from  the  so- 
called  Bacterium  pneumonia  crouposcz  in  that  it  is 
pathogenic  in  rabbits,  it  can  be  directly  isolated 
from  rusty  sputum,  and  it  requires  a  temperature 
for  its  growth  at  which  nutrient  gelatine  is  liquefied. 
The  cocci  were  first  observed  in  the  blood  of  a 
rabbit  inoculated  with  healthy  saliva,  and  again 
found  in  a  rabbit  which  died  after  inoculation  with 
the  saliva  of  a  child  suffering  from  rabies.  Later 
they  were  isolated  from  the  blood  of  rabbits  in- 
oculated with  the  buccal  secretions  of  different 
individuals,  and  were  found  to  be  constantly 
present  in  the  rusty  sputum  of  pneumonic  patients. 

Bacterium  crassum  sputigenum,  Kreibohm. 
—Short  thick  rods  with  rounded  ends.  Colonies 
on  plate-cultivations  appear  as  clear  grey-white 
points,  which  ultimately  form  greyish  slimy  drops. 
In  test-tubes  they  develop  very  quickly  a  nail- 
shaped  growth.  They  are  fatal  to  mice,  and  after 
death  are  found  in  the  blood,  and  in  sections,  more 
especially  in  the  capillaries  of  the  liver.  Rabbits 
die  of  septicaemia  after  intravenous  injection.  A 
large  quantity  of  a  cultivation  injected  into  the  cir- 
culation sets  up  fatal  gastro-enteritis  in  rabbits  and 
dogs  in  3  — 10  hours.  They  were  isolated  from 
sputum 

Bacterium  pneumonicum  agile  (Bacillus 
pneumonicm  agilis,  Schou.). — Short  thick  rods,  or 


238  BACTERIOLOGY. 

almost  elliptical  cells,  often  two  to  four  linked 
together.  They  form  dark  granular  colonies, 
which  after  twenty-four  hours  commence  to  liquefy 
the  gelatine ;  a  movement  is  then  visible  in  the 
centre  of  the  colony,  and  an  appearance  of  circum- 
ferential rays  results.  They  grow  also  on  blood 
serum,  bouillon,  and  potatoes.  Cultures  injected 
through  the  chest  wall,  or  into  the  trachea,  or  ad- 
ministered by  inhalation,  set  up  pneumonia.  They 
were  isolated  from  pneumonic  lungs  of  rabbit. 

Bacterium  oxytocum  perniciosum  (Bacillus 
oxytocus  perniciosus,  Wyssokowitsch). — Short  rods 
with  rounded  ends,  somewhat  shorter  and  thicker 
than  the  bacterium  of  sour-milk.  They  form 
yellowish  colonies,  and  in  test-tubes  develop  a  nail- 
shaped  growth.  Cultivated  in  milk  they  produced 
curdling,  and  an  acid  reaction.  They  were  some- 
times pathogenic  in  rabbits.  Isolated  from  sour 
milk. 

Bacterium  cavicida  (Bacillus  cavicida, 
Brieger). — Very  small  rods,  about  twice  as  long 
as  broad.  They  form  colonies  in  the  form  of 
whitish  concentric  rings.  On  potatoes  they 
develop  dirty  yellow  tufts.  They  are  very  fatal 
to  guinea-pigs.  Isolated  from  human  faeces. 

Bacterium  coli  commune,  Escherich. — Short, 
slightly-curved  rods,  1*5  /u,  in  length,  '3 — -4  thick, 
colonies  yellowish  and  granular.  They  develop 
a  white  scum  on  agar-agar  and  blood  serum. 
Fatal  to  guinea-pigs  and  rabbits,  when  inoculated 


SYSTEMATIC    AND    DESCRIPTIVE.  239 

intravenously.      Isolated  from  faeces  of  infants  fed 
exclusively  on  mothers'  milk. 

Bacterium  lactis  aerogenes,  Escherich.— 
Short  rods  with  rounded  ends,  1*4 — 2  /*,  long,  *5»/u, 
wide.  Cultivations  in  gelatine  resemble  the  bac- 
terium of  pneumonia.  They  produce  fermentation 
in  milk  and  in  solution  of  grape-sugar.  Patho- 
genic effects  similar  to  the  above.  Isolated  from 
the  same  source. 

Panhistophyton  ovatum,  Lebert  (Nosema 
bombycis,  Micrococcus  ovatus,  Corpuscles  du  ver  a  soie). 
— Shining  oval  cocci,  2 — 3  p,  long,  2  p.  wide,  singly 
and  in  pairs,  or  masses;  *  or  rods,  2-5  //,  thick,  and 
twice  as  long.f  They  multiply  by  subdivision. 
They  were  experimentally  proved  to  be  the  cause 
oi pd brine,  gat  tine,  maladie  des  corpuscles  Q?  Fiecksucht ; 
and  were  discovered  in  the  organs  of  diseased  silk- 
worms, as  well  as  in  the  pupae,  moths,  and  eggs. 

Bacterium  hyacinth!,  Wakker. — Cells  resem- 
bling Bacttrium  termo.  Observed  in  the  yellow 
slime  of  diseased  hyacinth  bulbs. 

Bacterium  synxanthum,Ehrenberg  (Bacterium 
xanthmum.  Bacterium  of  yellow  milk). — Cocci  "j — 
I  p.  in  length,  and  rod-forms. J  They  produce  a 
yellow  colour  in  boiled  milk,  which  at  first  becomes 
acid,  and  then  strongly  alkaline.  They  also  occur 
on  boiled  potatoes,  carrots,  etc.,  where  they  form 
small  lemon-yellow  masses.  The  colouring  matter 

*  Fliigge,  Fermente  und  Mikro-Parasiten.     1883. 
t  Zopf,  Die  Spaltpilze. 
t  Zopf,  Die  Sfialtyilze. 


240  BACTERIOLOGY. 

soluble  in  water,  insoluble  in  ether  and  alcohol,  un- 
changed by  alkalies,  decolorised  by  acids.  It  is 
similar  to  yellow  aniline  colours  both  spectro- 
scopically  and  in  ordinary  reactions. 

Bacterium  indicum  (Micrococcus  indicus,  Koch. 
Bacillus  indicus,  Fliigge). — Very  short  rods  with 
rounded  ends.  In  plate-cultivations  on  nutrient 
agar-agar,  the  colonies  have  a  scarlet  tint.  They 
are  round,  ovoid,  or  spindle-shaped,  and  have 
characteristic  granular  margins  (Fig.  82).  Grown 


FIG.  82. — BACTERIUM  INDICUM  ;  COLONIES  ON  NUTRIENT 
AGAR-AGAR,  x  60. 

upon  nutrient  agar-agar  in  a  test-tube,  the  appear- 
ances are  very  characteristic.  In  a  pure  cultivation 
a  brilliant,  vermilion-coloured  reticulated  pellicle 
develops  on  the  surface  (Plate  II.,  Fig.  i).  In  the 
track  of  the  needle  beneath  the  surface  no  pig- 
ment is  formed  (Plate  XIII.,  Fig.  2).  Cultivated 
in  nutrient  gelatine  they  liquefy  the  medium,  and 
colour  it  crimson.  The  growth,  of  a  darker 
crimson  hue,  subsides  to  the  bottom  of  the  tube. 
Upon  sterilised  potato  they  form  a  vermilion  layer 
(Plate  XV.,  Fig.  2). 


SYSTEMATIC    AND    DESCRIPTIVE.  24! 

Bacterium  rubrum,  Frank. — Minute  motile 
rods,  singly,  in  twos,  and  fours.  They  were 
observed  on  boiled  rice,  where  they  develop  a 
brick-red  pigment. 

Bacterium  prodigiosum  (Micrococcus  prodigi- 
osus,  Bacillus  prodigiosus*  "Blood-rain"  "Bleeding 
host"}. — Very  short  rods  with  rounded  ends,  and 
thread- forms,  '5 — i  /i  in  width,  forming  at  first 
rose-red,  and  then  blood-red  zoogloea.  They  grow 
luxuriantly  when  cultivated  on  sterilised  potatoes 
(Plate  IX.,  Fig.  i),  and  on  the  sloping  surface  of 
nutrient  agar-agar  (Plate  II.,  Fig.  3).  They 
appear  occasionally  on  bread,  boiled  rice,  and 
starch-paste,  and  more  rarely  on  boiled  white  of 
egg  and  meat.  Milk  sometimes  becomes  coloured 
blood-red  by  the  growth  of  this  fungus,  an  appear- 
ance formerly  attributed  to  a  disease  of  the  cow. 

In  Paris,  in  1843,  tne  fungus  was  peculiarly 
prevalent,  attacking  especially  the  bread  produced 
in  the  military  bakehouses. 

The  cells  themselves  are  colourless.  The 
colouring  matter  resembles  fuchsine;  it  is  in- 
soluble in  water,  but  soluble  in  alcohol.  The 
addition  of  acids  changes  it  to  a  carmine  red,  and. 
of  alkalies  to  a  yellow  colour. 

Bacterium  luteum  (Bacillus  lutem,  Fliigge). — 
Short  immotile  rods.  Colonies  irregular  in  form, 
appear  brownish  under  a  low  power,  but  macro- 
scopically  yellow.  In  test-tube  cultivations  they 
form  a  yellow  growth  without  liquefying  the 

16 


242  BACTERIOLOGY. 

gelatine.      They  occur   contaminating   plate-culti- 
vations. 

Bacterium  violaceum,  Bergonzini.  —  Cells 
similar  to  Bacterium  termo,  '6 — i  ^  thick,  2 — 3  p, 
long.  They  occur  on  white  of  egg,  forming  a 
yiolet  pigment. 

Bacterium  brunneum,  Schroter.  —  Motile 
rods,  producing  a  brown  colour.  They  were  ob- 
served on  a  rotting  infusion  of  maize. 

Bacterium  fluorescens  putidum  (Bacillus 
fluorescent  putidus,  Fliigge). — Short  rods  with 
rounded  ends  ;  motile  ;  spore-formation  not  known. 
They  form  small  dark  colonies  with  a  greenish 
sheen  and  penetrating  odour.  In  test-tubes  they 
produce  a  pale-grey  turbidity,  and  after  three  days 
colour  the  medium  with  a  greenish  tinge  spread- 
ing down  from  above.  On  potatoes  they  rapidly 
develop  a  brownish  layer.  They  occur  on  de- 
composing substances,  producing  a  greenish 
coloration. 

Bacterium  fluorescens  liquefaciens  (Bacil- 
lus fluorescens  liquefaciens,  Fliigge). — Short  rods  with 
rounded  ends.  Colonies  on  plates  develop  an  iri- 
descence around  them.  In  test-tubes  a  similar 
iridescent  sheen  is  produced.  On  potatoes  they 
develop  a  brownish  layer. 

.Bacterium  urese  (Micrococcus  urea,  Cohn). — 
Cocci  1*25 — 2  /A  in  diam.,  singly  or  in  chains,  and 
rods.  The  rods  split  up  by  division  into  chains  of 
cocci,  and  the  latter  are  finally  set  free.  The  cocci 


SYSTEMATIC    AND    DESCRIPTIVE.  243 

increase  further  by  subdivision,  and  a  jelly-like 
membrane  develops  around  them.  Masses  of  cocci 
exist  in  the  form  of  irregular  or  roundish  lumps. 
Cultivations,  after  twenty-four  hours,  consist  exclu- 
sively of  rods ;  after  forty-eight  hours,  of  cocci 
chains  ;  and  in  fourteen  days,  of  zoogloea  ;  the  cocci 
transplanted  into  fresh  nourishing  solution  again 
grow  into  rods.  These  observations  point  to  the 
existence  of  a  pleomorphic  species,  Bacterium 
urece,  and  the  former  nomenclature  Micrococcus 
urece  must  be  regarded  as  untenable.  They  are 
aerobic ;  occurring  in  urine  they  set  up  ammo- 
niacal  fermentation,  converting  urea  into  carbonate 
of  ammonia.*  Rods,  2  //,  long  and  i  p,  wide,  have 
been  isolated  from  stale  urine  (Bacillus  urece,  Leube), 
which  also  most  energetically  cause  the  ammoniacal 
fermentation  of  urine. 

Bacterium  aceti. — Cocci,  short  rods,  long  rods, 
leptothrix-forms,  and  zoogloea.  Cocci  and  short 
rods  may  occur  in  the  same  thread.  The  long 
rods  and  threads  may  develop  irregular  swellings, 
so-called  involution-forms,  which  have  a  thickened 
membrane  and  a  grey  colour.  The  effect  of  the 
action  of  this  microbe  is  to  oxidise  alcohol  in  wine 
and  other  fruit  juices  into  vinegar.  The  masses  of 
zooglcea  united  together  form  a  membranous  layer 
which  must  not  be  mistaken  for  the  pellicle  formed 
by  Saccharomyces  mycoderma.  The  latter  prepares  the 
medium  for  the  action  of  the  Bacterium  aceti. 
*  Zopf,  Die  Spaltyilze.  1885. 


244  BACTERIOLOGY. 

Bacterium  Pasteurianum,  Hansen.  Morpho- 
logically similar  to  Bacterium  aceti,  but  the  cells 
contain  a  starch-like  substance,  which  is  turned  blue 
by  iodine.  They  occur  in  beer-wort. 

Bacterium  liodermos  (Bacillus  liodermos, 
Fliigge.  Potato  bacterium).  —  Short  rods  with 
rounded  ends,  motile.  On  plate-cultivations  the 
colonies  appear  as  small  white  pellicles  floating 
on  liquefied  gelatine.  In  test-tubes  the  gelatine 
is  liquefied,  and  the  growth  sinks  down  in  floc- 
culent  masses.  They  occur  on  potatoes,  forming 
a  smooth  shining  layer,  which  ultimately  becomes 
crumpled. 

Bacterium  multipediculum  (Bacillus  multipedi- 
culus,  Fliigge). — Long  slender  rods.  They  form 
peculiar  insect-like  colonies  on  plate-cultivations. 
In  test-tubes  the  appearance  is  less  characteristic. 
They  occur  on  potatoes,  forming  a  dirty  yellow 
growth. 

Bacterium  ramosum  liquefaciens  (Bacillus 
ramosus  liquefaciens^  Fliigge). — Rods,  slowly  motile. 
They  form  characteristic  colonies  on  plate- 
cultivations.  The  colonies  gradually  sink  down, 
forming  a  well-marked  funnel  with  later  an 
appearance  of  concentric  rings.  In  test-tubes  the 
funnel-shaped  liquefaction  sends  off  rays  into  the 
surrounding  gelatine.  They  occur  occasionally, 
contaminating  cultivations. 

Bacterium  Zopfii,  Kurth. — Cocci,  i — 1-25  /*  in 
diameter  ;  rods,  and  threads.  Cultivated  in  a  streak 


SYSTEMATIC    AND    DESCRIPTIVE. 


245 


on  nutrient  gelatine  spread  out  on  a  glass  slide,  a 
peculiar  development  takes  place.  In  twenty-four 
hours  after  inoculation  threads  have  developed  ;  in 
forty-eight  hours,  windings  of  the  threads  are 
observed,  and  in  six  days  the  threads  have  broken 
up  into  cocci  (Fig.  83).  They  were  observed  in 


FIG.  83. — BACTERIUM  ZOPFII.  SUCCESSIVE  CHANGES  IN  THE  SAME 
THREAD,  X  740:  (a)  A  thread-form,  (b)  breaking  up  into  rod-forms, 
(c)  into  cocci  [after  Kurth]. 

the  intestine  of  fowls,  especially  in  the  contents  of 
the  vermiform  appendix.  Inoculation  of  rabbits  was 
followed  by  negative  results. 

Bacterium  merismopedioides,  Zopf. — Forms 
threads  i  — 1*5  p,  in  thickness;  these  subdivide  into 
long  rods,  short  rods,  and  finally  into  cocci.  The 


246  BACTERIOLOGY. 

cocci  divide  first  in  one  and  subsequently  in  two 
directions,  forming  characteristic  groups,  which 
appear  like  merismopedia.  These  groups  may 
eventually  consist  of  64  x  64  cells  or  more,  and 
ultimately  form  zoogloea.  The  cocci  develop  again 
into  rods  and  threads.  They  were  observed  in  water 
containing  putrefying  substances  (River  Panke, 
Berlin).* 

Bacterium  Pfliigeri,  Ludwig. — Large,  round 
cocci,  mostly  in  zooglcea,  and  thread-forms  com- 
posed of  rods.  They  can  be  cultivated  on  boiled 
white  of  egg  and  potatoes.  They  were  observed  to 
produce  phosphorescence  in  putrid  fish  and  meat. 

Bacterium  photometricum,  Engelmann. — 
Cells  slightly  reddish  in  colour,  motile.  The 
movements  are  stated  to  depend  on  light. 

Bacterium  litoreum,  Warming.— Cells  ellip- 
soidal 2 — 6  p,  long,  1*2 — 2 -4  ju,  wide,  occur  singly 
in  sea  water,  never  as  chains  or  zooglcea. 

Bacterium  fusiforme,Warming. — Cells  spindle- 
shaped,  with  pointed  ends,  2:5  /*  long  and  '5 — '8  p, 
thick.  Observed  as  a  spongy  layer  on  the  surface 
of  sea  water. 

Bacterium  navicula,  Reinke  and  Berthold. — 
Cells  spindle-form  or  ellipsoidal,  including  motile 
and  non -motile  forms.  They  have  one  or  more 
dark  spots,  which  may  be  coloured  blue  by  iodine. 
They  have  been  observed  in  rotting  potatoes. 

Proteus  vulgaris. — This  and  the  two  following 

*  Zopf,  Die  Spaltpilze.     1885. 


SYSTEMATIC    AND    DESCRIPTIVE.  247 

species  have  been  isolated*  from  putrefying  meat 
infusion,  and  are  stated  to  be  intimately  connected 
with  the  process  of  putrefaction.  In  the  history 
of  their  development  coccoid,  bacterioid,  spindle- 
form,  spirulinar,  and  involution  forms  have  been 
described.  In  Proteus  vulgaris  the  bacteria  vary  in 
size ;  some  measure  4  /x  in  length,  and  are  almost 
as  broad  as  long,  and  others  vary  from  "94 — 1*25  p, 
long  and  '42 — "63  wide.  They  are  actively  motile, 
and  cultivated  on  nutrient  gelatine  they  convert  it 
into  a  turbid,  greyish-white  liquid.  If  cultivated  in 
a  capsule  containing  5  per  cent,  of  nutrient  gelatine, 
a  few  hours  after  inoculation  the  most  characteristic 
movements  of  the  individual  bacilli  are  observed 
on  the  surface  of  the  nutrient  gelatine,  although  at 
this  early  stage  no  superficial  liquefaction  can  be 
detected.  Probably  the  movements  depend  upon 
the  existence  of  a  thin  liquid  layer,  as  they  are  not 
observed  if  the  nutrient  medium  contains  10  per 
cent,  of  gelatine. 

Proteus  mirabilis. — Cocci  -4  JLL — -9  /x,.  They 
occur  singly  and  in  zoogloea,  and  sometimes  in 
tetrads,  pairs,  chains,  or  as  short  rods  in  twos 
resembling  Bacterium  termo,  in  fact,  in  all  con- 
ceivable transition-forms.  Cultivated  on  nutrient 
gelatine  they  form  a  thick,  whitish  layer  in  con- 
centric circles,  which  in  time  liquefies  the  medium. 
Similar  movements  are  observed  in  capsule-cultiva- 
tions as  in  Proteus  vulgaris. 

*  Hauser,  Ueber  Faulniss-Bacterien.     1885. 


248  BACTERIOLOGY. 

Proteus  Zenkeri. — Cocci,  -4  ^  in  twos  like 
Bacterium  termo,  and  short  rods  1*65  ju,  long.  Cul- 
tivated on  nutrient  gelatine  no  liquefaction  results, 
but  a  thick,  whitish-grey  layer  is  formed.  The 
bacilli  are  motile,  and  the  same  phenomena  are 
observed  on  the  solid  medium  as  in  the  other  forms. 
In  cover-glass  impressions  most  varied  groupings 
of  the  bacilli  are  seen,  and  also  developmental  and 
involution-forms. 

The  two  following  forms  are  only  provisionally  re- 
garded as  distinct  species.  They  are  both  probably 
phase-forms  of  protean  species. 

Bacterium  termo,  Dujardin. — Short  cylindrical 
or  oblong  cells,  1*5  /*  long,  '5 — •/  broad,  generally  occurring 
as  dumb-bells.  The  cells  have  dark  contents,  invested  by 
a  thick  membrane,  and  are  provided  with  flagella,  to  which 
the  characteristic  movements  are  due  (Plate  I.,  Fig.  8). 
They  are  associated  with  putrefaction,  invariably  appear- 
ing in  decomposing  albuminous  substances  and  liquids.  A 
growth  can  be  readily  started  by  placing  a  piece  of  meat 
in  water  in  a  warm  place.  Cultivated  in  broth,  they  pro- 
duce a  turbidity,  and  on  sterilised  potatoes  a  slimy  grey 
layer. 

Bacterium  lineola.— Cells  3-8  /z— 5-2  //,  long,  1-5  //, 
wide.  They  occur  singly  or  in  pairs,  occasionally  in 
zooglcea,  but  never  in  chains.  The  cells  are  provided  with 
flagella,  and  contain  strongly  refringent  contents.  They 
resemble  Bacterium  termo  in  form  and  in  movement,  but 
are  considerably  larger.  They  occur  in  well  water  and 
stagnant  water,  and  form  slimy  heaps  on  rotting  potatoes, 
and  zoogloea  and  pellicles  on  various  infusions.  Cultivated 
on  nutrient  agar-agar  they  form  a  semi-transparent  growth 
(Plate  XXIV.,  Fig.  i). 


SYSTEMATIC    AND    DESCRIPTIVE.  249 

Genus  77. — Spirillum. 

SPECIES. 

ASSOCIATED    WITH   DISEASE  : — 

Spirillum  Obermeieri          .    Pathogenic. 

{Pathogenic  in  mnn  (?),  pos- 
sibly only  saprophytic. 
Pathogenic  in  animals. 

Spirillum  Finkleri  .  |  Saprophytic     in     man. 

I      Pathogenic  in  animals. 
In  animals        .         . !  Spirillum  tyrogenum  .         .  f  Saprophytic.      Pathogenic 
( Spirillum  sputigenum.  j      in  animals. 

UNASSOCIATED  WITH  DISEASE  : — 

Spirillum  plicatile 
Spirillum  serpens 
Spirillum  tenue  . 
Spirillum  undula 


Spirillum  volutans 
Spirillum  Rosenbergii 
Spirillum  attenuatum  . 
Spirillum  leucomelaneum 


Simple  saprophytes, 


Spirillum  Obermeieri  (Spiroch&te  Obermeieri, 
Cohn.  Spirillum  of  Relapsing  Fever}. — Threads 
similar  to  the  Spirillum  plicatile.  In  length  mostly 
1 6 — 40 /x,  with  screw-curves  regular  (Plate  I.,  Fig.. 
19).  They  move  very  rapidly,  and  exhibit  peculiar 
wave-like  undulations.  They  have  been  observed 
in  the  blood  of  patients  suffering  from  relapsing 
fever,*  but  never  in  the  secretions.  They  only  occur 
during  the  relapses,  and  are  absent  during  the  non- 
febrile  intervals.  Their  number  is  variable,  but 
usually  is  strikingly  great.  Outside  the  body,  in 
blood  serum  and  50  per  cent,  salt  solution,  the 
threads  preserve  their  movements.  From  analogy 
to  the  Spirillum  plicatile  it  is  presumed  that  these 
threads  are  composed  of  articulated  rods  and  cocci. 
Monkeys  have  been  inoculated  with  success  from 

*  Obermeier,  Med.  Centralb.     1873. 


250  BACTERIOLOGY. 

man,*  but  inoculations  of  mice,  rabbits,  sheep,  and 
pigs  gave  negative  results. 

The  spirilla  were  found  in  the  blood  of  the  in- 
oculated monkeys  in  great  numbers,  and  also  in  the 
brain,  lung,  liver,  kidney,  spleen,  and  skin  ;  and 
are  believed  to  be  the  cause  of  the  disease. 

METHODS   OF    STAINING   THE    SPIRILLUM  OBERMEIERI. 

In  cover-glass  preparations  of  blood  the  spirilla  stain 
strongly  with  fuchsine,  methyl-violet,  gentian-violet,  or 
bismarck-brown. 

In  sections,  brown  aniline  stains  have  been  recommended. 

Spirillum  cholerae  Asiaticae  (Comma-bacillus , 
Koch). — Curved  rods,  spirilla,  and  threads  (Plate  I., 
Fig.  1 8).  The  curved  rods  or  commas  are  about 
half  the  length  of  a  tubercle-bacillus.  They  occur 
isolated,  or  attached  to  each  other  forming  S-shaped 
organisms  or  longer  screw-forms  ;  the  latter  resem- 
bling the  spirilla  of  relapsing  fever.  Finally  they 
may  develop  into  spirilliform  threads.  In  old  cul- 
tivations threads  are  found  with  bulgings  or  irregu- 
larities, which  are  called  involution-forms  (Plate  I., 
Fig.  35). t  The  commas  are  actively  motile  ;  their 
movements  and  development  into  spirilla  may 
be  studied  in  drop-cultivations  (Fig.  84).  In  plate- 
cultivations,  at  a  temperature  of  from  16° — 20°  C.; 
the  colonies  develop  as  little  specks  (Fig.  85),  which 

*  Carter,  Lancet.     1879  and  1880.     Koch,  Co/fin's  Beitrdge. 
t  Compare  also  Van  Ermengem,  Recherches  stir  le  Microbe  du 
Choi.  Asiat.     1885. 


SYSTEMATIC   AND   DESCRIPTIVE.  251 

begin  to  be  visible  after  about  twenty-four  hours. 
Examined  with  a  low-power,  and  a  small  diaphragm, 
these  colonies  have  the  following  characteristics. 
They  appear  as  little  masses,  granular,  and  of  a 


FIG.  84.— COVER-GLASS  PREPARATION  OF  THE  EDGE  OF  A  DROP  OF  MEAT 
INFUSION,  containing  a  pure  cultivation  of  comma  bacilli,  with  (^a)  spirilli- 
form  threads,  X  600  [after  Koch]. 


very  faintly  yellowish-red  tinge,  which  have  lique- 
fied the  gelatine,  and  sunk  down  to  the  bottom 
of  the  resulting  excavations  (Fig.  86). 


FIG.  85.— COLONIES  OF  COMMA  BACILLI  ON  NUTRIENT  GELATINE, 
NATURAL  SIZE  [after  Koch]. 


In  test-tubes  of  slightly  alkaline  nutrient  gelatine 
(10  per  cent.),  the  appearance  of  the  growth 
is  very  striking.  It  commences  to  be  visible  in 


252 


BACTERIOLOGY. 


about  twenty-four  hours.     Liquefaction  sets  in  very 
slowly,    commencing    at    the   top     of   the    needle 


FIG.  86.— COLONIES  OF  KOCH'S  COMMA  BACILLI,   X   60  ;  from  a  nutrient 
gelatine  plate-cultivation. 

track  around  an  enclosed  bubble  of  air,  and  form- 
ing a  funnel  continuous  with  the  lower  part  of  the 


'    1JL 


Fig.  87. 


<f,£W 

k-f&. 


.'f* 


Fig.  88. 


FIG.  87.  —COVER-GLASS  PREPARATION  FROM  THE  CONTENTS  OF  A  CHOLERA 
INTESTINE,  x  600.  (a)  Remains  of  the  epithelial  cells  ;  (b)  Comma 
bacillus  ;  (^r)  Group  of  comma  bacilli  [after  Koch]. 

FIG.  88.  —  COVER-GLASS  PREPARATION  OF  CHOLERA  DEJECTA  IN  DAMP 
LINEN  (two  days  old),  X  600.  Great  proliferation  of  the  bacilli  with 
spirilla  (a)  [after  Koch]. 

growth   (Plate  III.,    Fig.    i)  ;  the  latter   preserves 
for    several     days    its    resemblance    to    a    white 


SYSTEMATIC    AND    DESCRIPTIVE. 


253 


thread  (Figs.  92  and  93).*  In  about  eight  days, 
however,  liquefaction  takes  place  along  the  whole 
of  the  needle  track. 

On  a  sloping  surface  of  agar-agar  the  cultivation 
develops  as  a  white,  semi-transparent  layer,  with  well- 
defined  margin.  In  potato-cultivations  the  microbe 


— — S 

M?   •*!'-.  *\9& 


FIG.  89. — SECTION  OF  THE  Mucous  MEMBRANE  OF  A  CHOLERA  INTESTINE, 
X  600.  A  tubular  gland  (a)  is  divided  transversely  ;  in  its  interior  (b) 
and  between  the  epithelium  and  the  basement  membrane  (r)  are  numerous 
comma  bacilli  [after  Koch]. 

will  only  grow  at  the  temperature  of  the  blood 
(37°C.),  forming  a  slightly  brown,  transparent  layer. 
Inoculation  of  a  cultivation  of  the  bacillus  in  the 
duodenum  of  guinea-pigs,  with  t  and  without  J 
ligation  of  the  bile  duct,  has  given  positive  results. 
More  recently  these  results  have  been  confirmed 
by  the  following  method.  Five  ccm.  of  a  5  per 

*  From  Remarks  on  the  Comma- Bacillus  of  Koch.  Lancet.  1885. 
t  Nicati  et  Rietsch,  Com.  a  I* Academic  de  Medecine.     1884. 
J  Van  Ermengem,  Le  Microbe  du  Cholera  Asiatique.     1885. 


254 


BACTERIOLOGY. 


cent,  solution  of  potash  were  injected  into  the 
stomach  of  a  guinea-pig,  and  twenty  minutes  after 
ten  ccm.  of  a  cultivation  of  comma-bacilli  diffused 
in  broth  were  similarly  introduced.  Simultaneously 

^"Jt^vSx 


Fig.  90.  Fig.  91.  Fig.  92.  Fig.  93. 

PURE  CULTIVATIONS  IN  NUTRIENT  GELATINE. 

Fig.  90.  Finkler's  bacillus,  twenty-four  hours  old. 
Fig.  91.         ,,  „        two  days  old. 

Fig.  92.  Koch's  cholera  bacillus,  twenty- four  hours  old. 
Fig.  93.          „  „  two  days  old. 

with  the  latter,  an  injection  of  tincture  of  opium 
was  made  into  the  abdominal  cavity,  in  the  propor- 
tion of  i  ccm.  for  every  200  grammes'  weight  of 
the  animal.  Those  who  have  had  success  with 


SYSTEMATIC    AND    DESCRIPTIVE.  255 

inoculation  experiments  maintain  that  choleraic 
symptoms  were  produced  without  any  trace  of 
peritonitis  or  putrid  infection,  and  that  the  comma- 
bacilli  of  Koch  were  again  found  in  the  intestinal 
contents,  and  fresh  cultivations  established. 

On  the  other  hand,  these  results  have  been  dis- 
puted, the  fatal  effects  of  the  inoculation  attributed 
to  septicsemic  poisoning,  and  the  proliferation  of 
the  bacilli  considered  to  be  dependent  upon  an 
abnormal  condition  of  the  intestines  induced  by  the 
injection  of  tincture  of  opium. *  It  is,  however,  very 
probable  that  these  organisms,  like  several  others 
which  have  been  isolated  from  intestinal  discharges, 
are  truly  pathogenic  in  the  lower  animals.  The 
comma-bacilli  were  found  in  the  superficial  necrosed 
layer  of  the  intestine,  in  the  mucous  flakes  and 
liquid  contents  of  the  intestinal  canal  of  cases  of 
Asiatic  cholera  t  (Figs.  87,  88,  89).  It  is  stated  that 
they  were  also  detected  in  a  tank  which  contained 
the  water  supply  to  a  neighbourhood  where  cholera 

*  Klein,  Brit.  Med.  Journal,  and  Micro-organism  and  Disease. 
1885.  Lankester,  Nature,  xxxi.;  Nineteenth  Century.  July,  1885, 
Klein  and  Gibbes,  "An  Inquiry  into  the  Etiology  of  Asiatic 
Cholera."  Bluebook,  1885. 

t  At  a  meeting  of  the  Physiological  Society,  May  I5th,  1886,  at 
Cambridge,  a  preliminary  communication  was  made  upon  the  investi- 
gations in  Spain  referred  to  in  the  first  edition  of  this  work.  The 
observations  made  by  Roy,  Brown,  and  Sherrington  rather  tend,  in 
the  opinion  of  the  author,  to  confirm  Koch's  views.  Comma-bacilli 
were  found  to  be  present,  in  some  cases,  in  enormous  numbers,  and 
the  frequency  of  their  occurrence  led  these  observers  to  believe  that 
they  must  bear  some  relation  to  the  disease.  At  the  same  time, 
as  they  failed  to  find  them  in  all  cases,  they  regarded  the  existence 
of  a  causal  relation  as  not  proven.  They  failed  to  find  the  Naples 
bacterium  or  the  small  straight  bacillus  noted  by  Klein ;  but  they 


256  BACTERIOLOGY. 

cases  occurred ;  but  comma-shaped  organisms  are 
commonly  present  in  sewage-contaminated  water 
(Fig.  94).  The  comma-bacilli  are  aerobic,  and 
their  development  is  arrested  by  deprivation  of 
oxygen.  They  are  destroyed 
by  drying  and  the  presence  of 
(,  C-  various  antiseptic  substances. 
/I  v  C  ^  They  are  distinguished  from 

i  I        '  J-Cj  "  all  other  comma-shaped  organ- 

isms by  the  test  of  cultivation. 
The    entirely   different    results 
obtained    in    the    case    of    the 
x  I200<  comma-bacilli  of  cholera  nostras 

(Figs.  90  to  93),  renders  a  thorough  acquaintance 
with  these  bacilli  of  the  greatest  importance  as  an 
aid  in  diagnosis. 

drew  attention  to  certain  peculiar  mycelium-like  threads  in  the 
mucous  membrane  of  the  intestines.  These  organisms,  however, 
judging*  from  a  preparation  stained  with  methylene  blue  which  was 
exhibited  at  the  meeting,  appeared  to  the  author  to  much  more 
closely  resemble  some  of  the  involution  forms  of  the  comma-bacillus, 
filaments  a  masses  globuleuses,  figured  by  Van  Ermengen,  than 
anything  else  he  had  seen.  Yet  assuming  these  peculiar  structures 
to  belong  as  described  to  some  species  of  Chytridiaceae,  it  is  very 
doubtful  whether  they  can  be  considered  to  have  any  significance. 
Methylene  blue  has  been  employed  by  Koch  and  others,  including 
the  author,  for  staining  sections  of  the  intestine  from  cholera  cases, 
and  had  they  been  constantly  present  it  is  hardly  possible  that  such 
striking  objects  could  have  been  overlooked.  Again,  we  must  bear 
in  mind  that  hyphomycetous  fungi  occasionally  have  been  found  to 
occur  saprophytically  in  the  intestinal  canal  as  well  as  in  the  lungs, 
external  auditory  meatus,  and  elsewhere.  We  must  wait,  before  ex- 
pressing a  more  definite  opinion,  until  the  report  of  these  observers 
is  published  in  full. 


SYSTEMATIC    AND    DESCRIPTIVE.  25) 

METHODS    OF    STAINING   THE    COMMA-BACILLI 

OF    KOCH. 

In  cover-glass  preparations  they  may  be  well  stained  in 
the  ordinary  way  with  an  aqueous  solution  of  methyl  violet 
or  fuchsine,  or  by  the  rapid  method,  without  passing  through 
the  flame  (p.  50,  Babes'  method). 
Nicati  and Rietsch's  method* 

A  small  quantity  of  the  stools  or  of  the  scraping  of  the 
intestinal  mucous  membrane  is  spread  out  on  a  glass 
slide  and  dried,  then  steeped  during  some  seconds 
in  sublimate  solution  or  in  osmic  acid  (i — 100).     It 
is   then   stained    by   immersion    in   fuchsine-aniline 
solution  (i  or  2  grammes  of  Bale  fuchsine  dissolved 
in  a  saturated  aqueous  solution  of  aniline,  washed, 
dried,  and  mounted  in  Canada  balsam. 
In   sections   of    the   intestine   their    presence    may   be 
demonstrated  by 
(a)  Koch's  method.\ 

Sections  of  the  intestine,  which  must  be  well  hardened 
in  absolute  alcohol,  are  left  for  twenty-four  hours  in 
a  strong  watery  solution  of  methylene  blue,  or  for  a 
shorter  time  if  the  colour  solution  is  wanned.  Then 
treated  in  the  usual  way. 
(ti)  Babes'  method. % 

Sections,  preferably  from  a  recent  case  of  cholera,  and 
made  as  soon  as  possible  after  death,  are  left  for 
twenty-four  hours  in  a  watery  solution  of  fuchsine 
(fabrique  de  Bale),  then  washed  in  distilled  water 
faintly  acidulated  with  acetic  acid,  or  in  sublimate 
solution  (i — 1000),  passed  rapidly  through  alcohol 
and  oil  of  cloves,  dried  with  filter  paper,  and  pre- 
served in  Canada  balsam. 

*  Brit.  Med.  Journal,  Sept.  1885. 

t  Berliner  Klinische  Woch.,  No.  31. 

|  Cornil  and  Babes,  Les  Bacteries,  p.  458.    1885. 

17 


2S8 


BACTERIOLOGY. 


Spirillum  Finkleri  (Comma- bacillus  in  Cholera 
nostras). — Curved  rods  thicker  than  the  comma- 
bacillus  of  Koch,  and  spirilla.  The  colonies  on 
plate-cultivations  (Plates  VI.  and  VII.)  are  very 
much  larger  than  those  of  the  comma-bacillus  of 

Koch  of  the  same  age. 
They  have  the  faintest 
yellowish-brown  tinge, 
a  well-defined  border, 
and  a  distinctly  granu- 
lar appearance.  They 
liquefy  nutrient  gela- 
tine very  rapidly,  so 
that  the  first  plate  of 
a  series  is,  as  a  rule, 
completely  liquefied 
on  the  day  following 
inoculation,  and  the 
second  plate  in  two  or 
three  days  more.  In  a 
test-tube  cultivation  in 
nutrient  gelatine  the 

Fig.  95.  Fig.  96.  appearances  are  espe- 

cially characteristic ; 
the  gelatine  is  very 
rapidly  liquefied  along 
the  whole  track  of  the 
needle,  so  that  the  cultivation  resembles  a  conical 
sack,  or  the  finger  of  a  glove  turned  inside  out 
(Figs.  95  and  96).  On  a  sloping  surface  of  nutrient 


PURE  CULTIVATIONS  OF  THE  SPIRILLUM 
FINKLERI  IN  NUTRIENT  GELATINE. 

Fig-  95-    In  twenty-four  hours. 
Fig.  96.    In  thirty-six  hours. 


SYSTEMATIC    AND    DESCRIPTIVE.  259 

agar-agar  a  white  moist  layer  forms  very  quickly. 
On  potatoes  they  grow  at  the  ordinary  temperature 
of  the  air,  producing  a  brownish  layer  and  corrosion 
of  the  surface  of  the  potato.  They  were  discovered 
in  the  evacuations  of  cases  of  cholera  nostras,  and 
were  claimed  at  first  to  be  identical  with  the 
comma-bacillus  of  Koch.  By  the  test  of  cultivation 
they  are  now  ascertained  to  be  distinct.  They  also 
have  been  shown  to  be  pathogenic.* 

Spirillum  sputigenum,   Lewis. — Curved   rods 
very  similar  to  the  comma-bacilli  of  Koch.      Many 


FIG.  97. — SPIRILLUM  SPUTIGENUM.  Occurring  with  spirochseta  denticola, 
leptothrix-filaments,  micrococci,  and  bacteria  in  a  scraping  from  a  carious 
tooth,  X  1200. 

have  failed  with  repeated  attempts  to  cultivate 
these  bacilli,  and,  therefore,  maintain  that  they 
are  quite  distinct  biologically  from  the  spirilla 
associated  with  Asiatic  cholera.  Others  assert 
that  they  can  be  cultivated  in  an  acid  nutrient 
gelatine,  and  that  they  are  identical  with  Koch's 
comma-bacilli  in  their  mode  of  growth.  They 
occur  with  other  bacteria  in  saliva,  and  in 
scrapings  from  carious  teeth  (Fig.  97). 

*  Finkler  and   Prior,    Erganzungshefte  zztm  Centralblatt  fur 
Allgemeine  Gesundheitspflege,  Erster  Band.  1885. 


26O  BACTERIOLOGY. 

Spirillum  tyrogenum,  Deneke. — Curved  rods, 
slightly  smaller  than  Koch's  comma-bacilli,  with 
a  great  tendency  to  form  long  spirillar  threads 
(Fig.  98).  The  colonies  on  plate-cultivations  are 
sharply  defined,  and  of  a  greenish-brown  colour. 
After  a  time  they  liquefy  the  gelatine,  but  the 
liquefaction  is  much  more  marked  than  in  colonies 
of  Koch's  commas  of  the  same  age,  though  not  so 
rapid  as  in  the  case  of  the  commas  of  cholera 
nostras.  In  test-tubes  of  nutrient  gelatine  a  turbid 
liquefaction  occurs  along  the  needle  track,  and  on 
the  surface  of  nutrient  agar-agar  a  yellowish-white 


FIG.  98.-  -SPIRILLUM  TYROGENUM.     From  a  cultivation  in  nutrient 
gelatine,  X    1200. 

layer  develops.  Inoculation  of  potatoes  gives  no 
result.  Administration  of  the  bacilli  by  the  mouth, 
in  the  manner  employed  for  testing  the  pathogenic 
effect  of  Koch's  bacilli,  produced  a  fatal  result  in 
a  few  cases ;  on  the  other  hand,  injection  into  the 
duodenum  failed  entirely.  The  pathogenic  proper- 
ties may  be,  therefore,  considered  as  not  yet 
established.  They  were  isolated  from  old  cheese. 
Spirillum  plicatile,  Ehrenberg  (Marsh-Spiro- 
chcete). — Thin  threads,  2*25  p  in  breadth,  with 
numerous  narrow  windings,  1 10 — 125  //.long,  occur- 
ring also  in  spirulinar  forms.  The  threads  have 
primary  and  secondary  windings  ;  the  former  are 
in  each  example  of  equal  size,  but  the  latter  are  often 


SYSTEMATIC    AND    DESCRIPTIVE.  26 1 

irregular;  their  ends  are  cut  off  bluntly,  and  they 
exhibit  rapid  movement.  They  occur  abundantly 
in  marsh-water  in  summer,  and  can  be  obtained 
by  allowing  algae  to  decompose  in  water  (Fig.  99). 
On  cultivation  the  threads  break  up  into  long  rods, 
short  rods,  and  finally  cocci.  This  change  is  ren- 
dered visible  by  making  cover-glass  preparations, 
and  staining  with  aniline  dyes. 


FIG.  99. — SPIRILLUM   PLICATILE   (Marsh   Spirochaete).      From  sewage- 
contaminated  water,  X  1200. 

The  following  may  be  provisionally  described  as  dis- 
tinct species ',  though  they  are  probably  the  spiral  phase- 
forms  of  protean  species. 

Spirillum  serpens,  Miiller  (Vibrio  serpens). — 
Threads  II—  28  //-  long,  *8 — i'i  fj,  thick,  with  three  or  four 
windings.  They  are  actively  motile,  often  united  into 
chains,  or  forming  swarms,  and  are  abundant  in  stagnant 
liquids. 

Spirillum  tenue. — Very  thin  threads,  with  at  least 
i^,  usually  2 — 5  spirals.  Height  of  a  single  screw  is 
2—3  p,  and  the  length  of  spiral,  therefore,  4 — 15  p.  They 
are  very  swiftly  motile,  and  often  occur  in  felted  dense 
swarms  in  vegetable  infusions. 

Spirillum  undula. — Threads  ri— 1-4  ^  thick,  9— 
12  fju  long;  spirals  4-5  //,  high;  each  thread  has  if— 3 
spirals.  They  are  actively  motile,  and  possess  at  each  end 
a  flagellum.  They  occur  in  various  infusions  (Fig.  100). 

Spirillum  volutans,  Ehrenberg. — Threads  1-5— 


2O2  BACTERIOLOGY. 

2  p  thick,  25 — 30  fju  long  ;  tapering  towards  their  extremi- 
ties, which  are  rounded  off.  They  possess  dark  granular 
contents.  Each  thread  has  24—3^  windings  or  spirals, 
whose  height  is  9—13  p.  They  have  a  flagellum  at  each 
end,  and  are  sometimes  motile,  sometimes  not.  They  are 
found  in  various  infusions  and  water  of  marshes. 

Spirillum  Rosenbergii. — Threads  with  i— ij 
windings;  4 — 12  /*  long;  1-5 — 2'6  p  thick.  They  are 
colourless,  but  the  contents  include  strongly  refractive 
sulphur  granules.  Also  spirals  6—7-5  P  in  height,  which 
are  actively  motile,  are  found  in  brackish  water. 


FIG.  100. —SPIRILLUM  UNDULA,  x  1500. 

Spirillum  attenuatum,  Warming. — Threads 
much  attenuated  at  the  ends,  which  consist  usually  of  three 
spirals.  The  middle  spiral  is  about  n  JJL  high,  and  6  p  in 
diameter  ;  and  the  end  ones  10  /u,  high,  and  2  /z-in  diameter. 
They  are  found  in  brackish  water. 

Spirillum  leucomelaneum,  Koch. — A  rare  form 
observed  in  water  covering  rotting  algae.  Dark  and  glass- 
like  spaces  alternate  in  the  spirillum,  resulting  from  a  regular 
arrangement  of  the  dark  granular  contents. 

Genus  IIL — Leuconostoc. 

SPECIES. 

UN  ASSOCIATED   WITH    DISEASE  : — 

Leuconostoc  mesenteroides     .     .     .     Zymogenic  saprophyte. 

Leuconostoc      mesenteroides,       Cienkowski 

(Gomme     de     sucrerie,     Froschlaichpilz,      Frogspawn 

fungus). — Cells  singly,  in  chains,  and  in   zoogloea, 


SYSTEMATIC    AND    DESCRIPTIVE. 


263 


surrounded  by  a  thick  gelatinous  envelope  (Fig. 
101).  The  life-history  has  been  very  thoroughly 
investigated.*  The  spores,  1*8 — 2  p  in  diameter, 
are  of  a  round  or  ellipsoidal  form,  with  thick  mem- 


FIG.  101. — LEUCONOSTOC  MESENTEROIDES. 

I.  Spores.  2.  Spores  after  germination,  showing  gelatinous  envelope. 
3,  4,  5,  6.  Increase  by  division.  7.  Glomerular  form  of  zooglcea. 
8.  Section  of  an  old  mass  of  zoogloea.  9.  Cocci  chains  with  arthro- 
spores  [after  Tieghem  and  Cienkowski]. 

brane  and  shining  contents.  The  outer  membrane- 
layer  bursts,  and  a  middle  lamella  oozes  out,  and 
forms  a  thick  gelatinous  envelope,  while  the  inner 
layer  remains  adherent  to  the  plasma.  Thus  the 
spore-germination  leads  to  the  formation  of  a 

*  Cienkowski,  Die  Gallertbildungen  d.  Zuckerrubensaftes.  1878; 
and  Van  Tieghem,  "  Stir  la  Gomme  de  ucrerie,"  Ann.  Sc.  Nat. 
1879. 


264  BACTERIOLOGY. 

coccus  with  a  gelatinous  envelope.  The  coccus 
then  elongates  into  a  short  rod-form,  and  the 
gelatinous  envelope  becomes  ellipsoidal.  The  rod 
divides  into  two  cocci,  and  each  of  these  lengthens 
into  a  rod  and  divides.  By  repetition  of  this  pro- 
cess a  chain  of  cocci  results,  encased  in  a  cylindri- 
cal or  ellipsoidal  envelope.  The  chains  increase  in 
length,  become  twisted  up,  and  eventually  fall 
apart  into  pieces  of  various  lengths.  In  nourish- 
ing liquids  a  great  number  of  little  masses  are 
formed,  which  adhere  together,  and  produce 
pseudo-parenchymatous  structures.  These  latter 
may  join  together,  forming  still  larger  agglomera- 
tions. The  masses  of  zooglcea  are  of  almost  a 
cartilaginous  consistency,  and  admit  of  sections 
being  made  with  a  razor.  After  a  long  time  the 
envelope  liquefies,  and  the  cocci  are  set  free  ;  the 
latter  introduced  into  fresh  nourishing  media  de- 
velop new  colonies.  In  the  chains  some  of  the 
cocci  become  enlarged  without  changing  their 
form.  These  acquire  the  properties  of  spores, 
and  are  called  arthro-spores  (p.  131). 

This  micro-organism  occurs  occasionally  in 
beet-root  juice  and  the  molasses  of  sugar-makers, 
forming  large  gelatinous  masses  resembling  frog- 
spawn.  The  vegetation  is  so  rapid  that  forty-nine 
hectolitres  of  molasses,  containing  10  per  cent,  of 
sugar,  were  converted  within  twelve  hours  into  a 
gelatinous  mass ;  consequently,  it  is  a  formidable 
enemy  of  the  sugar  manufacturers. 


SYSTEMATIC    AND    DESCRIPTIVE. 


Genus  IV. — Bacillus. 


SPECIES. 


ASSOCIATED  WITH  DISEASE  : — 
Bacillus  leprse 
Bacillus  in  syphilis 

Bacillus  typhosus 
Bacillus  malarioe  . 


In 


Pathogenic. 

Pathogenic  (?) ;  possibly  only 
saprophytic. 

Pathogenic  in  man  (?),  patho- 
genic in  animals. 

Pathogenic. 

Saprophytic  in  man,  patho- 
genic in  animals. 
Saprophytic. 

>  •> 
Pathogenic. 


In  animals 


Bacillus  of  choleraic  diarrhoea  from 

meat  poisoning 
Bacillus  pyogenes  foetidus 

Bacillus  in  septicaemia  in  man 
Bacillus  in  gangrenous  septicaemia 
Bacillus  tuberculosis 
Bacillus  anthracis 
Bacillus  mallei      .... 
Bacillus  of  malignant  oedema 
Bacillus  of  septicaemia  of  mice 
Bacillus  of  ulcerative  stomatitis  in 

the  calf 

Bacillus  of  swine-typhoid 
Bacillus  of  swine-erysipelas  . 
Bacillus  in  tetanus 
Bacillus  alvei        .... 

UNASSOCIATED  WITH  DISEASE  : — 
Bacillus  ianthinus 
Bacillus  pyocyaneus 
Bacillus  cyanogenus      .         .         .  ' 
Bacillus  acidi  lactici      .         .         .  | 
Bacillus  Fitzianus          .         .         .  ] 
Bacillus  subtilis 
Bacillus  fi^urans   . 
Bacillus  of  jequirity 
Bacillus  caucasicus 
Bacillus  dysodes   .... 
Bacillus  Hansenii 
Bacillus  erythrosporus  . 
Bacillus  septicus   .... 
Bacillus  saprogenes 
Bacillus  fcetidus    . 
Bacillus  putrificus  coli  . 
Bacillus  coprogenus  foetidus  . 
Bacillus  aerophilus 
Bacillus  mesentericus  fuscus . 
Bacillus  mesentericus  vulgatus 

Bacillus  leprae,  Hansen. — Fine  slender  rods, 
4 — 6  //,  long,  and  less  than  i  p  wide,  occasionally 
pointed  at  both  ends,  some  clearly  motile,  and 
others  not.  In  tissue  sections  they  have  a  beaded 


Pathogenic  (?) 
Pathogenic. 


Chromogenic  saprophytes. 
Zymogenic  saprophytes. 


Simple  saprophytes. 


266  BACTERIOLOGY. 

appearance  (Fig.  102).  Spore-formation  has  been 
described.  They  have  been  cultivated  artificially 
on  blood  serum  and  alkaline  meat  extract.  In- 
oculation experiments  on  monkeys  and  other 
animals  have  failed  to  produce  the  disease ;  though 
in  cats  and  rabbits  there  have  been  indications 
of  success.*  The  bacilli  occur  in  enormous  num- 
bers in  tubercular  leprosy  in  the  nodules  of  the 
skin  (Plate  XXIII.,  Figs,  i  and  2),  and  of  the 
mucous  membrane  of  the  mouth,  palate,  larynx, 


FIG.  102. — LEPROSY  BACILLI  FROM  A  SECTION  OF  SKIN,  x  1200. 

etc.f  They  occur  also  in  the  liver,  spleen,  testicles, 
lymphatic  glands,  and  kidneys  (Plate  XX.,  Fig.  2) ; 
and  in  the  interstitial  tissue  of  the  nerves  in 
anaesthetic  leprosy.  They  probably  spread  by 
the  lymphatics,  and  are  not  found  in  the  blood. 
In  their  behaviour  to  staining  reactions  they  are 
similar  to  the  bacillus  of  tubercle,  except  that  they 
stain  much  more  readily. 

METHODS    OF    STAINING     THE     BACILLUS    OF    LEPROSY. 

Cover-glass  preparations  may  be  made  in  the  ordinary 
way,  or  by  a  special  method,  which  consists  in  clamping  a 
nodule  with  a  pile-clamp,  until  a  state  of  anaemia  of  the 
tissue  is  produced.  On  pricking  with  a  needle  or  sharp 
knife  a  drop  of  clear  fluid  exudes,  from  which  cover-glass 

*Damsch,  Virchow's  Archiv,  Bd.  92,  Heft  i. 
tThin,  Med.   Chir.  Trans.  Lond.,  1883;  Brit.  Med.  Journal, 
No.  129,  1884,  and  Steven,  Brit.  Med.  Journal,  No.  1281,  1885. 


SYSTEMATIC    AND    DESCRIPTIVE.  26/ 

preparations  may  be  made.*  Cover-glass  preparations 
and  sections  may  be  stained  by  Ehrlich's  method  (p.  50), 
or  the  latter  by  the  following  process : — 

Method  of  Babes.  \ — Preparations  are  stained  in  a  solution 
of  rosaniline  hydrochlorate  in  aniline-water.  Decolorise 
in  33  per  cent,  hydrochloric  acid,  and  after-stain  with 
methylene  blue. 

Bacillus  in  syphilis,  Lustgarten.J — Rods  re- 
sembling the  bacilli  of  leprosy  and  tuberculosis, 
3 — 4  fJL  long,  *8  p  thick.  Two  or  more  colourless, 
ovoid  points  in  the  course  of  the  rod  are  visible  with 
a  high  power ;  it  is  thought  that  they  are  possibly 
spores.  The  bacilli  are  always  found  in  the  interior 
of  nucleated  cells  which  are  more  than  double  the 
size  of  leucocytes.  They  have  been  observed  in  the 
discharge  of  the  primary  lesion,  and  in  hereditary 
affections  of  tertiary  gummata.  Some  observers 
state  that  an  identical  bacillus  is  found  in  normal 
secretions,  §  and  others  ||  have  described  a  bacillus 
associated  with  specific  lesions,  which  is  stated  to 
differ  from  the  above  in  its  behaviour  towards  stain- 
ing reagents. 

METHOD    OF    STAINING    THE    BACILLUS   OF    SYPHILIS. 

Method  of  Lust  gar  ten  : — 

Sections  are  placed  for  from  twelve  to  twenty-four  hours 
in  the  following  solution,  at  the  ordinary  temperature  of 

*  Manson,  Lancet.     1884. 

t  Babes,  Compt.  Rend,  de  V Acad.  d.  Sc.    1883. 

|  Lustgarten,  Die  Syphilisbacillen.     Mit  4  Tafeln.    1885. 

§  Alvarez  et  Tavel,  "  Recherches  sur  le  Bacille  de  Lustgarten," 
Archiv  de  Phys.  Norm,  et  Path.,  17  ;  Klemperer,  "  Ueber  Syphilis 
und  Smegma  Bacillen,"  Deutsche  Med.  Woch.  1885. 

||  Eves  and  Lingard,  Lancet,  April  loth,  1886. 


268  BACTERIOLOGY. 

the  room,  and  finally  the  solution  is  warmed  for  two  hours 

at  60°  C.  :— 

Concentrated  alcoholic  solution  of  gentian-violet  .      1 1 
Aniline  water        *  *       .         .         .         .         .,.100 

The  sections  are  then  placed  for  a  few  minutes  in 
absolute  alcohol,  and  from  this  transferred  to  1-5  per  cent, 
solution  of  permanganate  of  potash.  After  ten  minutes 
they  are  immersed  for  a  moment  in  a  pure  concentrated 
solution  of  sulphurous  acid.  If  the  section  is  not  com- 
pletely decolorised,  immersion  in  the  alcohol  and  in  the 
acid  bath  must  be  repeated  three  or  four  times.  The 
sections  are  finally  dehydrated  with  absolute  alcohol, 
cleared  with  clove-oil,  and  mounted  in  Canada  balsam. 

Method  of  De  Giacomi  : — 

Cover-glass  preparations  are  stained  with  hot  solution 
of  fuchsine  containing  a  few  drops  of  perchloride  of  iron. 
They  are  then  decolorised  in  strong  perchloride  of  iron 
and  after-stained  with  vesuvin  or  bismarck  brown. 

Method  of  Doutrelepont  and  Schiitz  : — 

Sections  are  stained  in  a  weak  aqueous  solution  of 
gentian  violet  and  after-stained  with  safranin. 

Bacillus  typhosus,  Eberth  (Bacillus  in  typhoid 
fever). — Rods,  '2  p,  broad,  and  forming  filaments 
up  to  50  JJL  long ;  *  ort  rods,  short,  rounded  at  their 
ends,  and  occasionally  constricted  in  the  middle ; 
some  exhibiting  spore-formation.  These  bacilli 
have  been  observed  in  inflamed  Peyer's  glands,  in 
the  spleen,  mesenteric  glands,  and  the  lungs  in 
fatal  cases  of  typhoid  fever.  More  recently  {  a 
bacillus  has  been  cultivated  on  several  plates  of 

*  Kleb's  Arch.  f.  Experimental  Pathol.     1880. 

t  Eberth,  Virchovfs  Archiv,  Bd.  83. 

\  Gaffky,  Mittheil.  a.  d.  K.  Gesundheitsamte.     1884. 


SYSTEMATIC    AND    DESCRIPTIVE.  269 

gelatine  which  were  inoculated  from  different 
spleens.  After  twenty-four  hours  the  course  of  the 
inoculation  streak  became  visible,  and  in  forty-eight 
hours  a  distinct  whitish  growth  had  developed.  With 
a  low  power  this  was  found  to  consist  of  numerous 
colonies  of  a  yellow-brownish  colour.  The  gelatine 
was  not  liquefied.  The  rods  varied  in  length 


FIG.  103.— BACILLUS  TYPHOSUS  FROM  A  POTATO-CULTIVATION,  x  1500. 

(Fig.  103),  were  capable  of  development  into  threads, 
and  were  motile.  They  can  be  cultivated  on  potatoes 
at  37°  C.  They  grow  well  also  on  blood  serum, 
forming  a  whitish-grey,  somewhat  transparent  layer. 
Spore-formation  occurs  at  the  ends  of  the  rods.  It 
is  stated  that  inoculation  experiments  have  been 
made  in  some  cases  with  success.* 

METHODS    OF    STAINING   THE    BACILLUS    OF    TYPHOID 

FEVER. 

To  stain  the  bacilli  in  the  tissues  the  method  of  Gram 
can  be  employed,  or  the  sections  may  be  left  for  twenty- 
four  hours  in  methylene-blue.  Koch  recommends  bis- 
marck-brown.  To  colour  the  spores  cover-glass  preparations 
and  sections  must  be  left  for  several  days  in  the  fuchsine 
solution  employed  in  the  method  of  Ehrlich  (p.  163) ;  or 
the  solution  may  be  warmed,  and  in  the  case  of  cover- 
glasses,  even  raised  to  boiling-point.  They  are  then 
decolorised  with  nitric  acid,  and  after-stained  with 
methylene  blue. 

*  Fraenkel  and  Simmonds,  Die  Atiolog.  bedeutung  des  Typhus  - 
bacillus.  1886. 


27O  BACTERIOLOGY. 

Bacillus  malarise,  Klebs  (Bacillus  in  inter- 
mittent fever}. — Rods,  2 — 7  /x,  long,  which  grow 
into  twisted  threads.  Spore-formation  takes  place 
in  the  centre,  or  at  either  end  (Plate  I.,  Fig.  14). 

Inoculated  in  rabbits  they  were  stated  to  produce 
a  febrile  disorder  analogous  to  malarial  fever,* 
and  in  the  spleen  and  marrow  the  threads  and 
spores  of  the  bacilli  were  found  in  abundance. 
Bacilli  with  end-spores  have  been  discovered  also 
in  the  blood  of  patients  suffering  from  malaria. t  , 

The  bacilli  were  first  described  as  present  in  the 
soil  of  the  Roman  Campagna. 

According  to  more  recent  observations,  peculiar 
amoeboid  bodies  and  motile  filaments  are  constantly 
present  in  the  blood  of  cases  of  malaria.  These 
organisms,  or  plasmodia  malaria,  appear  to  be  closely 
allied  to  the  flagellated  protozoa  (see  Appendix  B). 

Bacillus  of  choleraic  diarrhoea  from  meat- 
poisoning,  Klein.  J — Rods  from  3 — 9  /i  in  length, 
i -3  /i  wide,  rounded  at  their  extremities,  singly 
or  in  chains  of  two.  Spore-formation  occurs,  the 
spores  being  i  ^  thick,  oval,  and  situated  in  the 
centre  or  at  the  end  of  the  rod. 

Feeding  with  the  bacilli  and  inoculation  produced 
positive  results.  At  the  autopsy,  pneumonia,  peri- 
tonitis, pleuritis,  enlargement  of  the  liver  and  spleen, 
and  haemorrhage  were  observed,  and  bacilli  were 

*  Klebs  and  Tommasi  Crudeli,  Archiv.  f.  Experimental  Pathol. 
1879. 

t  Marchiafava,  ibid. 
\   Klein,  p.  87. 


SYSTEMATIC    AND    DESCRIPTIVE.  27! 

present  in  the  blood  and  exudations  of  the  animal. 
They  occurred  in  the  blood  and  juices,  and  espe- 
cially in  the  glomeruli  of  the  kidneys,  of  several 
fatal  cases  of  choleraic  diarrhoea. 

Bacillus  pyogenes  fcetidus,  Passet. — Small 
rods,  with  rounded  ends  of  about  i  '45  p,  in  length, 
and  '58  p,  in  width  ;  often  in  twos,  or  linked 
together  in  chains.  They  are  motile,  and  spore- 
formation  occurs.  When  cultivated  in  nutrient 
gelatine,  a  greyish,  veil-like  growth  forms  on  the 
surface.  In  plate  cultivations  white  points  appear 
after  twenty-four  hours,  which  develop  into  greyish 
spots,  and  these  enlarging  coalesce  into  a  layer. 
In  nutrient  agar-agar  the  cultivation  resembles  the 
growth  on  gelatine.  On  blood  serum  a  moderately 
thick  greyish-white  streak  develops,  and  on  steril- 
ised potato  an  abundant,  shining,  brownish  culture. 
From  all  these  media  a  putrid  odour  emanates,  but 
no  smell  is  detected  from  a  cultivation  in  milk. 
Inoculated  into  mice  and  guinea-pigs  abscesses 
are  produced  or  death  from  septicaemia  results. 
They  were  isolated  from  putrid  pus. 

Bacillus  in   septicaemia   of  man,   Klein.*— 
Rods   singly  or  in  chains,  I — 2*5  p,  long,    3 — "5  p. 
wide,  which  were  observed  in  the  blood-vessels  of 
the  swollen  lymphatic  glands.     They  are  possibly 
identical  with  the  following:  — 

Bacillus  in  gangrenous  septicaemia,  Ar- 
loing  and  Chauveau.  —  Short  rods,  possessing 

*  Klein,  Micro-organisms  and  Disease.     1885. 


272  BACTERIOLOGY. 

spores,  were  observed  around  wounds  in  gangre- 
nous septicaemia,  and  considered  to  be  the  cause 
of  the  gangrene. 

Bacillus  tuberculosis,  Koch.* — Rods,  2 — 4  p 
and  occasionally  8  p,  long,  very  thin,  and  rounded 
at  the  ends.  They  are  straight  or  curved,  and 
frequently  beaded  (Fig.  104),  and  occur  singly,  in 
pairs,  or  in  bundles.  They  are  found  in  the  cells 


FIG.  104.— BACILLUS  TUBERCULOSIS,  FROM  TUBERCULAR  SPUTUM,  STAINED 
BY  EHRLICH'S  METHOD,  X  2500.     From  photographs. 


of  tubercles,  especially  in  the  interior  of  giant 
cells.  In  the  latter  they  are  often  accompanied 
with  grains  which  exhibit  the  same  colour  reaction 
(Plate  XVIII. ,  Fig.  i).  They  are  non-motile. 
Spore-formation  has  been  described  (see  p.  134). 
The  best  medium  for  cultivation  is  solid  blood 
serum  of  cow  or  sheep,  with  or  without  the 
addition  of  gelatine  ;  and  the  most  favourable  tem- 
perature for  their  development  is  37° — 38°  C.  The 

*  Koch,  Berl.  Klin.  Woch.,  No.  15,  1882  ;  and  MittheiL  aus  dem 
Kaiser  lich.  Gesundheitsamte,  "  ^tiologie  der  Tuberkulose." 


SYSTEMATIC    AND    DESCRIPTIVE.  273 

growth  takes  place  very  slowly,  and  only  between 
the  temperatures  of  30°  and  41°  C.  In  about  eight 
or  ten  days  the  growth  appears  as  little  whitish  or 
yellowish  scales  and  grains  (Plate  XL,  Fig.  i). 

The  bacillus  can  also  be  cultivated  in  a  glass 
capsule  on  blood  serum,  and  the  appearances  of 
the  growth  studied  under  the  microscope.  The 
scales  or  pellicles  are  then  seen  to  be  made  up  of 
colonies  of  a  perfectly  characteristic  appearance, 
which  may  be  still  further  studied  by  making  a 
cover-glass  impression  (p.  52,  and  Plate  XL,  Fig. 
4).  They  are  then  seen  to  be  composed  of  bacilli, 
arranged  more  or  less  with  their  long  axis  corre- 
sponding with  that  of  the  colony  itself,  and  with  an 
appreciable  interval  between  the  individual  bacilli. 
The  colonies  themselves  appear  as  fine  curved  lines, 
the  smallest  being  mostly  S-shaped.  Longer 
colonies  have  serpentine  twistings  and  bendings, 
which  often  recall  the  curves  of  fancy  lettering.  The 
ends  of  the  lines  run  to  sharp  points,  but  the  middle 
of  the  growth  is  spindle-formed.  The  youngest 
colonies  are  extremely  delicate  and  narrow,  but  the 
older  colonies  increase  in  size,  are  thicker  across, 
and,  blending  with  each  other,  gradually  obliterate 
the  characteristic  appearances  ;  a  lamellated  growth 
results,  which  increases,  and  gives  the  appearance 
to  the  naked  eye  of  the  scale  or  pellicle  already 
described.  The  blood  serum  is  not  liquefied  unless 
putrefactive  bacteria  contaminate  the  culture.  A 

fresh  tube  can  be  inoculated  with  one  of  these  little 

18 


274  BACTERIOLOGY. 

scales,  and  a  new  generation  started.  The  scales 
gradually  increase  in  size,  and  consist  entirely  of 
bacilli.  In  about  three  to  four  weeks  the  cultivation 
ceases  to  increase,  and  it  is  then  necessary  to 
inoculate  a  fresh  tube.  The  virulence  is  not 
weakened  by  carrying  on  successive  cultivations. 
A  relatively  small  portion  of  the  cultivation  inocu- 
lated into  the  subcutaneous  tissue,  into  the  peritoneal 
or  pleural  cavity,  into  the  anterior  chamber  of  the 
eye,  or  directly  into  the  blood  stream,  produces 
after  three  or  more  weeks  artificial  tuberculosis  in 
guinea-pigs  and  rabbits.  Dogs  and  cats  can  also 
be  infected  by  experimental  inoculation. 

The  appearances  observed  at  the  autopsy  are, 
swollen  lymphatic  glands  in  the  neighbourhood  of 
the  inoculation;  followed  by  softening  and  abscess; 
enlargement  of  the  spleen  and  liver,  with  for- 
mation of  caseous  tubercles ;  tuberculosis  of  the 
lungs,  bronchial  glands,  and  peritoneum.  After 
inoculation  of  the  eye,  grey  tubercles  appear  on 
the  iris,  and  undergo  enlargement  and  caseation, 
followed  by  tuberculosis  of  the  eyeball  and  organs 
generally.  The  bacilli  appear  to  be  the  direct 
cause  of  tuberculosis,  and  the  presence  of  the 
bacillus  in  the  sputum  of  patients  is  regarded 
as  a  distinctive  sign  of  the  existence  of  this 
disease.  The  detection  of  the  bacillus  has,  con- 
sequently, become  a  test  which  is  daily  applied 
by  physicians  in  forming  clinical  diagnoses. 

The  bacilli  are  found  in   all  tubercular  growths 


SYSTEMATIC    AND    DESCRIPTIVE.  275 

of  man,  monkeys,  cattle  (Perhuchf),  birds,  and  many 
other  animals,  and  in  cases  of  artificial  tuberculosis, 
in  rabbits,  guinea-pigs,  cats,  etc.  (Plate  XVIIL,  Fig. 
2).  In  man  the  bacillus  can  be  detected  in  the  tis- 
sues, in  the  sputum,  in  the  blood,  and  in  the  urine.* 
Tuberculosis  may  also  be  produced  by  inhalation 
and  feeding  experiments  (p.  107).  The  channels  of 
infection  in  man  are  also  most  probably  the 
pulmonary  or  intestinal  mucous  membranes.  The 
possibility  of  inoculation  of  skin  wounds  is  open  to 
doubt.  The  bacilli  or  their  spores  are  inhaled  from 
the  air,  or  taken  in  with  food.  As  a  relatively  high 
temperature  is  required  for  their  growth,  they  cannot 
thrive  outside  the  animal  body  in  cold  climates. 
Morphologically  identical  bacilli  have  also  been 
observed,  but  very  sparsely,  in  sections  of  lupus. 

METHODS    OF    STAINING    THE    TUBERCLE    BACILLUS. 

Numerous  methods  have  been  recommended  for  staining 
the  bacillus  tuberculosis,  each  of  which  will  be  given  in  detail 

EhrlicJis  and  Gibbes  methods  may  be  with  advantage 
employed  in  staining  cover-glass  preparations.  Gibbet 
rapid  double  stain  is  obviously  the  best  to  employ  for 
clinical  purposes.  For  sections  both  EJirlicJis  and  Neehens 
methods  give  excellent  results. 

Koch's  original  method. — Cover-glass  preparations  or 
sections  are  laid  in  Koch's  solution  (No.  23,  c.)  for  twenty- 
four  hours,  or  for  one  hour  -if  the  solution  is  warmed  to 
40°  C.  Rinse  in  water ;  immerse  in  a  watery  solution 
of  vesuvin  for  two  minutes ;  rinse  again  in  water,  and 
examine  ;  or,  after  rinsing  in  water,  treat  with  alcohol, 
clove-oil,  and  Canada  balsam. 

*  Babes,  Centralbl.f.  d.  Med.  Wz'ssensch.,  1883,  p.  145. 


276  BACTERIOLOGY. 

x*.*-;    yf^/>  v 

EhrlicJis  method. — Cover-glass-preparations  are  allowed 
to  float  in  a  watch-glass,  containing  a  solution  of  gentian- 
violet  or  fuchsine,  added  to  aniline  water.  A  saturated 
alcoholic  solution  of  the  dye  is  added  till  precipitation 
commences  (10  ccm,  aniline  water,  and  10 — 20  drops  of 
the  colour  solution).  The  cover-glasses  are  left  in  the  solu- 
tion for  about  half  an  hour ;  then  washed  for  a  few  seconds 
in  strong  nitric  acid  (one  part  commercial  nitric  acid  to  two 
of  distilled  water),  and  rinsed  in  distilled  water.  After- 
stain  with  vesuvin  or  methylene-blue,  rinse  in  water,  dry 
and  preserve  in  Canada  balsam  (Plate  XX.,  Fig.  i). 

Sections  and  cover-glass-preparations  may  be  stained 
by  this  method,  as  described  by  Koch.* 

Saturated  alcoholic  solution  of  methyl-violet 

or  fuchsine 1 1 

Aniline  water        ......  loo 

Absolute  alcohol 10 

Preparations  are  left  for  twelve  hours  in  this  solution 
(colouring  of  the  cover-glass-preparations  can  be  expedited 
by  warming  the  solution). 

Treat  the  preparations  with  (i — 3)  solution  of  nitric  acid 
a  few  seconds. 

Wash  in  alcohol  (60  per  cent.)  for  a  few  minutes  (cover- 
glass-preparations  need  only  be  rinsed  a  few  times).  After- 
stain  with  diluted  solution  of  vesuvin  or  methylene-blue 
for  a  few  minutes. 

Wash  again  in  60  per  cent,  alcohol,  dehydrate  in  absolute 
alcohol.  Clear  with  cedar-oil,  mount  in  Canada  balsam. 

RindfleiscK s  method. — Prepare   a  solution   composed   of 

Saturated  alcoholic  solution  of  fuchsine     10  drops 
Aniline  water          .         .         .         .         .2  drams. 

Pour  it  into  a  watch-glass,  and  float  the  cover-glass ; 
warm  the  watch-glass  over  a  spirit-lamp  until  steam  rises. 
Remove  it  from  the  flame,  and  set  it  aside  for  five  minutes, 

*  Mittheil.  aus  dem  Gesundheitsamte,  Zweiter  Band,  1884,  p.  10 


SYSTEMATIC    AND    DESCRIPTIVE.  277 

Take  out  the  cover-glass,  and  transfer  it  for  a  few  seconds  to 
acidulated  alcohol  (two  drops  of  nitric  acid  in  a  watch-glass 
full  of  alcohol).  Wash  in  distilled  water,  dry,  and  preserve 
in  balsam.  After-stain,  if  necessary,  with  bismarck- 
brown,  or  methylene-blue. 

Weigert-Ehrlich  method  (vide  p.  61). 

Orttis  modification  of  Ehrlictis  method. — Stain  by  the 
method  of  Ehrlich,  but  decolorise  with  acidulated  alcohol 
(one  of  hydrochloric  to  one  hundred  parts  of  70  per  cent, 
alcohol). 

Gibbes*  method* — Stain  cover-glass-preparations  in  ma- 
genta solution  (No.  22)  for  15 — 20  minutes.  Wash  in 
(i — 3)  solution  of  nitric  acid,  until  the  colour  is  removed. 
Rinse  in  distilled  water.  After-stain  with  methylene- 
blue,  methyl-green,  iodine-green,  or  watery  solution  of 
chrysoidin,  five  minutes.  Wash  in  distilled  water  till  no 
more  colour  comes  away.  Transfer  to  absolute  alcohol 
for  five  minutes ;  dry,  and  preserve  in  Canada  balsam. 
Leave  sections  in  the  stain  for  half  an  hour,  then  treat 
with  nitric  acid,  and  wash  with  distilled  water.  Transfer 
to  methylene-blue  till  deeply  stained,  wash  again  in 
distilled  water,  and  then  in  spirit.  Pass  through  absolute 
alcohol  and  clove-oil,  and  preserve  in  Canada  balsam. 

Gibbes'  new  method. — Cover-glass-preparations  are  placed 
in  the  double  staining  solution  (No.  1 6),  which  has  been 
warmed  in  a  test-tube,  and,  as  soon  as  steam  rises,  poured 
into  a  watch-glass.  They  are  allowed  to  remain  for  five 
minutes,  and  then  are  washed  in  methylated  spirit  till  no 
more  colour  comes  away,  dried  in  the  air  or  over  a  spirit- 
lamp,  and  mounted  in  Canada  balsam.  If  the  solution  is 
used  without  warming,  the  cover-glasses  must  be  left  in  it 
for  an  hour.  Sections  are  treated  on  the  same  principles, 
but  must  be  left  in  the  solution  for  several  hours.  The 
crumpling  of  the  sections  by  the  action  of  nitric  acid  is 
avoided. 

*  Gibbes,  Practical  Pathology.     1 883 . 


278  BACTERIOLOGY. 

Baumgarttris  method. — Cover-glass-preparations  of  sputum 
are  made  as  already  described  (p.  48),  and  immersed  in 
a  very  dilute  solution  of  potash  (i — 2  drops  of  a  33  per 
cent,  solution  of  potash  in  a  watch-glass  of  distilled  water) 
The  cover-glass  is  pressed  down  on  a  slide,  and  examined 
with  a  high  power.  The  bacilli  can  be  thus  examined  in 
the  unstained  condition,  and  to  avoid  any  mistake  from 
confusion  with  other  species,  the  cover-glass  can  be  re- 
moved, dried,  passed  through  the  flame,  and  stained  with 
a  drop  of  an  aqueous  solution  of  fuchsine,  or  gentian-violet. 
The  putrefactive  bacteria  are  stained,  but  the  tubercle 
bacilli  remain  absolutely  colourless. 

Baumgartens  new  method. — A  solution  is  prepared  as 
follows  :  Drop  4 — 5  drops  of  concentrated  alcoholic  methyl- 
violet  solution  into  a  small  watch-glass  full  of  water. 
(a)  Stain  the  sections  in  this  solution,  wash  them  in  water,  and 
decolorise  in  absolute  alcohol  (five  to  ten  minutes),  or,  before 
treating  with  alcohol,  immerse  the  sections  for  five  minutes 
in  a  half-saturated  solution  of  carbonate  of  potash.  Pass 
through  clove-oil,  and  mount  in  a  mixture  of  Canada  balsam, 
free  from  chloroform,  and  clove-oil  (equal  parts).  The  object 
of  this  process  is  to  differentiate  the  tubercle  bacilli  from 
chance  bacteria,  inasmuch  as  the  tubercle  bacilli  gradually 
are  decolorised  by  the  clove-oil,  (b)  Sections  stained  in 
the  above  solution  are  placed  for  five  minutes  in  alcohol, 
and  then  in  a  concentrated  solution  of  bismarck-brown  in 
I  per  cent,  solution  of  acetic  acid.  The  after-treatment 
may  be  conducted  as  already  described. 

Neelsen's  method.  —  Cover-glass-preparations  may  be 
quickly  stained  in  Neelsen's  solution  (No.  2$)  warmed  in 
a  watch-glass  till  steam  rises.  Sections  are  left  for  from 
five  to  ten  minutes  in  the  solution,  and  then  washed  in  a 
watery  solution  of  sulphuric  acid  (25  per  cent.)  ;  rinsed  in 
distilled  water,  and  immersed  in  methylene  blue  solution. 
After  two  or  three  minutes  they  are  passed  through  alcohol 
and  oil  of  cloves,  and  mounted  in  Canada  balsam. 


SYSTEMATIC    AND    DESCRIPTIVE.  279 

Balmer •-  Frantzel  method.  —  Dissolve  two  grammes  of 
freshly-powdered  gentian-violet  in  100  grammes  of  aniline- 
water.  Immerse  the  sections  for  twenty-four  hours,  and 
treat  as  in  Ehrlich's  method. 

ZiehVs  method. — Stain  with  Ehrlich's  method,  but  omit 
the  nitric  acid ;  after-stain  with  methylene-blue.  The 
latter  replaces  the  stain  of  all  bacteria  except  the  tubercle 
bacillus. 

Lichtheims  method. — Concentrated  solution  of  fuchsine 
or  gentian-violet  is  diluted  with  distilled  water,  and  the 
sections  stained  for  thirty-six  hours. 

Peters  method. — Sections  are  stained  for  half  an  hour  in 
fresh  aniline-gentian-violet  solution.  Transfer  to  20  ccm. 
of  absolute  alcohol  for  eighteen  hours,  the  alcohol  being 
renewed  two  or  three  times.  Rinse  in  distilled  water  for 
one  minute,  and  immerse  for  three  minutes  in  a  watery 
solution  of  aniline-yellow  (aniline-yellow  '2  dissolved  in 
distilled  water  10,  filter).  Wash  in  absolute  alcohol,  clarify 
with  clove-oil,  and  preserve  in  Canada  balsam. 

Franker s  method. — Sputum  preparations  are  rapidly 
double-stained  by  the  following  method :  Prepare  a  solu- 
tion by  adding  concentrated  alcoholic  methyl-violet  or 
fuchsine  solution,  drop  by  drop  till  opalescence  arises,  to 
5  ccm.  of  aniline-water  heated  to  100°  C.  Float  the 
prepared  cover-glasses  two  minutes  in  the  warmed  solu- 
tion. The  process  of  after-staining  and  decolorisation  is 
effected  by  placing  the  preparation  for  one  to  two  minutes 
in  one  of  the  following  solutions :  for  fuchsine-stained 
preparations  a  saturated  solution  of  methylene-blue  in  a 
mixture  of 

Alcohol 50 

Distilled  water     ....         30 
Nitric  acid 20 

which  is  filtered  before  use ;    for  preparations  stained  in 
methyl-violet,  a  saturated  solution  of  vesuvin  may  be  used  in 


2  80  BACTERIOLOGY. 

Alcohol         .....        7° 
Nitric  acid    ...  .30 

which  must  be  filtered  before  use.  The  sections  are 
washed  in  water  (or  weakly  acidified  50  per  cent,  alcohol), 
dried  and  mounted  in  the  usual  way. 

Pfuhl-Petris  method. — The  colouring  solution  consists  of 
10  ccm.  of  a  saturated  alcoholic  solution  of  fuchsine  added 
to  100  ccm.  of  water.  Float  the  cover-glasses  for  two 
minutes  in  the  solution  heated  till  steam  rises.  Wash  for 
one  minute  in  glacial  acetic  acid,  rinse  in  water,  and  after- 
stain  in  an  alcoholic  or  watery  solution  of  malachite  green 
for  a  half  or  one  minute.  Rinse  again  in  water.  Dry,  and 
examine  in  glycerine,  or  preserve  in  Canada  balsam. 

SenkewitscK s  method. — Stain  cover-glass-preparations  in 
concentrated  fuchsine  solution.  When  strongly  coloured, 
wash  out  the  stain  for  one  to  two  minutes  in  alcohol,  to 
which  one  drop  of  nitric  acid  has  been  added  for  every 
10  ccm.  Rinse  in  water,  dry,  and  mount  in  Canada  balsam. 

Kaatzers  method. — Place  the  cover-glass-preparations 
for  twenty-four  hours  in  a  solution  of  over-saturated  alco- 
holic gentian-violet,  or,  if  warmed  to  80°  C.,  for  three 
minutes.  Decolorise  in  a  solution  consisting  of 

Alcohol  90  per  cent.         .         .         .         100  ccm. 
Water    . *  ,         .         .         .  20  ccm. 

Strong  hydrochloric  acid  .         .  20  drops. 

Rinse  in  90  per  cent,  alcohol,  and  after-stain  with  concen- 
trated watery  solution  of  vesuvin  for  two  minutes  ;  wash 
again  in  distilled  water,  dry,  and  mount  in  Canada  balsam. 
EhrlicJis  method  and  eosin. — The  author  has  found  that 
after  sections  have  been  stained  with  methyl-violet  and 
bismarck-brown  by  Ehrlich's  method,  as  described  by  Koch 
(p.  163),  they  may  with  advantage  be  immersed  in  a  weak 
alcoholic  solution  of  eosin,  then  rinsed  in  clean  absolute 
alcohol,  clarified  with  clove-oil,  and  mounted  in  Canada 


SYSTEMATIC    AND    DESCRIPTIVE.  28 1 

balsam.  The  giant  cells  are  then  stained  pink,  while 
their  nuclei  are  brown  and  the  bacilli  blue  (Plate  XVIII., 
Fig.  i). 

Bacillus  anthracis  (BacUridie  du  charbon,  Bacil- 
lus of  splenic  fever,  woohorters*  disease,  or  malignant 
pustule). — Rods,  5 — 20  p  long  and  i — 1*25  /x  broad, 
and  threads  ;  spore-formation  present.  As  a 
thorough  knowledge  of  the  life-history  of  this 
bacillus  is  of  the  greatest  importance,  inasmuch  as 
it  is  without  any  doubt  the  actual  cause  of  wide- 
spread disease,  the  various  steps  to  be  followed 
in  a  practical  study  of  it  will  be  successively  treated 
in  detail.  Its  morphological  and  biological  charac- 
teristics have  been  very  completely  worked  out, 
and  it  serves  as  an  excellent  subject  for  gaining 
an  acquaintance  with  the  various  methods  that 
should  be  employed  in  studying  micro-organisms. 
It  is  found  that  a  mouse  inoculated  with  the  bacillus 
or  its  spores  will  die  in  from  twenty-four  to  forty- 
eight  hours,  or  more  rarely  in  from  forty-eight  to 
about  sixty  hours. 

Examination  after  death. — The  details  to  be  ob- 
served in  the  autopsy  have  already  been  described 
(p.  in).  The  spleen  is  found  to  be  consider- 
ably enlarged,  and  may  be  removed  (p.  112),  and 
examined  by  making  cover-glass  preparations, 
inoculations,  and  subsequently  sections. 

Cover-glass  preparations* — In  cover-glass  prepara- 
tions of  the  blood  of  the  spleen  the  bacilli  are  found 
in  enormous  numbers.  Preparations  should  be 


282  BACTERIOLOGY. 

made  similarly  with  blood  from  the  heart  and  exuda- 
tions from  the  lungs,  etc.  In  the  last-mentioned  the 
bacilli  are  present  in  very  small  numbers,  or  alto- 
gether absent.  They  should  be  examined  both  un- 


FIG.  105.— BACILLUS  ANTHRACIS,  x  1200.  From  a  preparation  of  blood  from 
the  spleen  of  an  inoculated  mouse. 


stained  and  stained  (p.  48).  The  rods  are  straight, 
or  sometimes  curved,  rigid,  and  motionless,  and  vary 
in  size  in  different  animals.  They  stain  intensely 
with  aniline  dyes,  and  are  then  seen  to  be  composed 
of  segments  with  their  extremities  truncated  at  right 


i 


FIG.  1 06.— BACILLUS  ANTHRACIS,  X  1200.  From  a  cover-glass  preparation 
stained  with  Gram's  method  and  eosin.  The  light  part  of  the  rod 
represents  the  sheath  stained  pink,  and  the  dark  part  the  protoplasmic 
contents  stained  blue  and  contracted. 

angles  ;  between  the  segments  a  clear  linear  space 
exists,  which  gives  them  a  characteristic  appearance 
(Fig.  105).  By  double  staining  (p.  49),  the  rods  are 
seen  to  consist  of  a  membrane  or  hyaline  sheath 
with  protoplasmic  contents  (Fig.  106). 


SYSTEMATIC   AND   DESCRIPTIVE.  283 

Drop-cultures. — A  little  of  the  blood  from  the 
spleen  or  heart  is  employed  to  inoculate  the  liquid 
medium,  bouillon  or  blood  serum.  Several  of 
these  cultures  should  be  prepared,  and  some  of 
them  placed  in  the  incubator.  Examined  from 
time  to  time  it  will  then  be  observed  that  the  rods 
grow  into  long  homogeneous  filaments,  which  are 
twisted  up  in  strands,  and  then  untwisted  in  long 
and  graceful  curves.  In  a  few  hours  they  begin  to 
swell,  become  faintly  granular,  and  finally,  bright, 
oval  spores  develop  (Plate  I.,  Fig.  28).  The  cul- 
tures in  the  incubator  develop  rapidly,  a  tempera- 
ture of  25° — 40°  C.  being  most  favourable  for 
the  growth  of  the  bacillus.  The  spores  are 
eventually  set  free,  and  by  making  a  fresh  cul- 
tivation, or  by  injecting  them  into  a  mouse  or 
guinea-pig,  they  germinate  again  into  the  cha- 
racteristic bacilli,  which  in  their  turn  grow  into 
filaments  and  spores.  When  the  spore  germinates 
it  swells,  the  outer  layer  becomes  jelly-like,  and 
giving  way  at  one  or  other  pole,  the  contents 
escape  and  grow  into  a  rod.  With  the  precautions 
previously  described  (p,  112)  cultivations  should 
be  established  in  nutrient  gelatine,  nutrient  agar- 
agar,  and  on  sterilised  potatoes. 

Test-tube  cultivations  in  nutrient  gelatine . — Typically 
characteristic  appearances  are  obtained  by  inocu- 
lating a  5  to  8  per  cent,  nutrient  gelatine.  A 
whitish  line  develops  in  the  track  of  the  inoculating 
needle,  and  from  it  fine  filaments  spread  out  in  the 


284 


BACTERIOLOGY. 


gelatine  *  (Fig.  107).  Occasionally  a  little  isolated 
spot  develops,  from  which  rays  extend  in  all  direc- 
tions, like  the  silky  filaments  of  thistle-down. 
The  filaments  are  more  easily  observed  with  a 
magnifying  glass.  In  a  more  solid  nutrient- 
gelatine  the  growth  appears  only  as  a  thick 
white  thread.  As  liquefaction  of  the  gelatine 
progresses,  these  appearances  rapidly 
disappear,  and  the  growth  subsides  as 
a  white  flocculent  mass  (Plate  V.,  Fig. 
3).  In  exhausted  culture-media,  and 
sometimes  in  the  blood,  filaments  are 
seen  in  a  state  of  degeneration.  This 
has  also  been  observed  in  sections  of 
the  kidney,  etc.,  of  a  rabbit  inoculated 
with  the  anthrax  bacillus,  which  had 
died  of  septicaemia  the  following 
morning. 

Test-tube  cultivations  in  nutrient  agar- 
agar. — Cultivated  upon  a  sloping  sur- 
face  of  nutrient    agar-agar   a   viscous 
snow-white   layer    is    developed  (Plate 
FuRiTcuLTivA-  XIV.,  Fig.  i).     Without  access  of  air 

TION  OF  THE  ...  ,  ,          .  ,  . 

BACILLUS  AN-  no    cultivation    can    be    obtained,    the 

THRACIS  IN         n  .,,...  .    .  rj^,    .  , 

NUTRIENT     bacilli   being    serobic.       Ihis    can     be 

NK     demonstrated   by   embedding    a   piece 

of  lung  or  spleen  pulp  containing  bacilli  in  nutrient 

agar-agar  (p.  137).     No  growth  of  the  bacilli  takes 

place. 

*  The  Author,  Lancet,     1885. 


SYSTEMATIC    AND    DESCRIPTIVE.  285 

Potato -cultivations. — A  very  characteristic  growth 
results  from  the  inoculation  of  sterilised  potatoes. 
The  damp-chamber  containing  the  potatoes  is 
placed  in  the  incubator,  and  in  about  thirty-six 
to  forty-eight  hours  a  creamy,  very  faintly  yellow- 
ish layer  forms  over  the  inoculated  surface,  with 
usually  a  peculiar  translucent  edge  (Plate  XV., 
Fig.  i).  On  removing  the  cover  of  the  damp- 
chamber  a  strong,  penetrating  odour  of  sour  milk 
is  encountered. 


FIG.  108.— COLONIES  IN  A  PLATE-CULTIVATION,  x  70. 

Plate-cultivations. — From  the  spleen  or  blood  of 
the  heart,  cultivations  must  be  established  in 
nutrient  gelatine  on  plates.  The  colonies  develop 
in  about  two  days,  according  to  the  temperature  of 
the  room.  They  appear  to  the  naked  eye  as  little 
white  spots  or  specks,  which,  on  examination  with 
a  low  power  of  the  microscope  and  small  diaphragm, 
exhibit  two  distinct  forms.  One  form,  on  careful 
focussing,  has  the  appearance  of  a  little  compact 
ball  of  twisted  thread  ;  in  the  other,  liquefaction 
of  the  gelatine  has  commenced,  and  the  thread 


286  BACTERIOLOGY. 

bundles  are  spreading"  out  like  locks  or  plaits  of 
hair  in  the  neighbouring  gelatine.  These  appear- 
ances are  perfectly  characteristic  (Fig.  108). 

Cover- glass  impressions.  —  The  plate-cultivations 
should  be  also  examined  as  soon  as  the  colonies 
appear,  by  making  cover-glass  impressions  (p.  52), 
and  staining  them  with  aniline  dyes.  The  filaments, 
examined  with  a  high  power,  will  then  be  seen  to 
consist  of  a  number  of  rods  or  segments  (Plate  I., 
Fig.  30).  On  the  other  hand,  filaments  from  a 


FIG.   109.— COVER- GLASS  IMPRESSION-PREPARATION,  X  70. 

tube  cultivation  in  a  solid  medium  will  be  found 
to  be  composed,  not  only  of  rods,  but  here  and 
there  of  torula-like  in  volution- forms  (Plate  I.,  Fig. 
30).  In  a  cover-glass  impression  from  a  potato - 
culture  (Plate  I.,  Fig.  29)  the  individual  segments 
have  a  great  tendency  to  be  isolated  one  from  the 
other,  and  there  is  copious  spore-formation. 

Preservation  of  spores. — Spores  may  be  preserved 
simply  by  allowing  anthracic  blood  to  dry  and 
sealing  it  in  a  tube.  The  spores  from  a  potato 


SYSTEMATIC    AND    DESCRIPTIVE.  287 

cultivation  are  treated  as  follows : — The  inocu- 
lated surface  containing  the  creamy  cultivation 
is  sliced  off  in  a  thin  layer,  and  is  mashed  up 
with  distilled  water  in  a  glass  capsule.  Sterilised 
silk-thread  is  cut  up  into  lengths  of  about  a  quarter 
of  an  inch,  and  allowed  to  soak  in  the  paste  for 
some  hours,  under  a  bell-glass.  The  threads  are 
then  picked  out  with  a  pair  of  forceps,  and  laid  upon 
a  sterilised  glass  plate,  covered  with  a  bell-glass, 
and  allowed  to  dry.  From  the  plate,  when  perfectly 
dry,  they  are  transferred  to  a  small  test-tube,  which 
can  be  plugged  with  cotton -wool,  or  sealed  in  the 
Bunsen  burner. 

Examination  in  the  tissues. — The  organs  must  be 
hardened  in  absolute  alcohol,  cut  and  stained, 
(pp.  54,  291).  The  method  of  Gram  is  the  most 
instructive,  and  eosin  a  very  satisfactory  contrast 
stain.  The  capillaries  all  over  the  body,  lungs, 
liver,  kidney,  spleen,  skin,  mucous  membrane,  etc., 
will  be  found  to  contain  bacilli.  In  some  cases  the 
bacilli  are  so  numerous  (t-g.-,  in  the  capillaries  of 
the  kidney,  Plate  XVI.,  Fig.  2),  that  examination 
with  a  low  power  gives  the  appearance  of  an 
injected  specimen. 

Inoculation  of  animals. — A  thread  containing  spores, 
a  drop  of  blood  from  an  infected  animal,  or  a 
minute  portion  of  a  cultivation,  introduced  under 
the  skin  of  a  mouse  or  guinea-pig,  causes  its  death, 
as  a  rule,  in  from  twenty-four  to  forty-eight  hours. 
Sheep  fed  upon  potatoes  which  have  been  the 


288  BACTERIOLOGY. 

medium  for  cultivating  the  bacillus,  die  in  a  few 
days.  Goats,  hedgehogs,  sparrows,  cows,  horses, 
are  all  susceptible.  Rats  are  infected  with  difficulty. 
Pigs,  dogs,  cats,  white  rats,  and  Algerian  sheep 
have  an  immunity  from  the  disease.  Frogs  and 
fish  have  been  rendered  susceptible  by  raising  the 
temperature  of  the  water  in  which  they  lived. 

Dissemination  of  the  disease  and  mode  of  infection. — It 
has  been  stated  that  when  carcases  of  animals  which 
have  died  of  anthrax  are  buried  under  the  soil,  the 
development  of  the  bacilli  into  spores  can  take  place. 
The  spores  were  supposed  to  be  taken  up  by  earth 
worms,  carried  to  the  surface,  and  deposited  in  their 
castings  ;  animals  then  grazing  or  sojourning  on 
the  soil  are  thus  liable  to  be  infected.*  This  has  not 
been  borne  out  by  experiment^  Bacilli,  however, 
occur  in  large  numbers  in  the  blood  and  discharges 
from  the  nose  and  mouth  of  the  moribund  animals, 
and  in  the  urine  and  faeces.  They  find  a  nourishing 
soil  in  decaying  vegetable  and  animal  matter,  and 
having  free  access  of  oxygen  form  copious  spores, 
so  that  the  grass  is  extensively  contaminated. 

In  warm  and  marshy  districts  the  spore-formation 
is  still  more  active,  and  the  spores  may  be  carried 
by  floods  over  adjacent  meadows.  As  to  the  mode 
of  infection,  the  animals  may  be  directly  infected 
through  buccal  wounds  caused  by  siliceous  grasses, 
or  by  wounds  of  insects  ;  the  intestinal  and  pul- 

*  Pasteur,  Bulletin  de  VAcademie  de  Medecine.     1880. 
t  Koch,  Mittheil.  a.d.  Gesundheitsamte.     1881 


SYSTEMATIC    AND    DESCRIPTIVE.  28$ 

monary  mucous  membranes  are  also  regarded  as 
pathways  of  infection.  In  animals  the  disease  is 
known  as  "  splenic  fever." 

In  man  the  mode  of  infection  is  by  inhalation  of 
spores,  and  ingress  by  the  pulmonary  or  intestinal 
mucous  membrane,  or  by  direct  inoculation  of  a 
wound  or  abrasion.  The  spores  are  derived  from 
the  wool  or  hides  of  animals  which  have  died  of 
anthrax,  and  the  resulting  disease  is  known  as 
"  wool- sorter's  disease,"  and  "  pustula  maligna." 
Bacilli  are  found  in  the  serum  of  the  pustule,  and  in 
sputum,  urine,  faeces,  and  sweat.;  and  if  the  disease 
prove  fatal,  in  the  capillaries  throughout  the  body. 

Attenuation  of  the  virus. — By  cultivating  the 
bacillus  in  neutralised  bouillon  at  42° — 43°  C.  for 
about  twenty  days,  the  infecting  power  is  weakened, 
and  animals  inoculated  with  it  {premier  vacciri)  are 
protected  against  the  disease.*  To  obtain  a  still 
more  perfect  immunity,  they  are  inoculated  a  second 
time  with  material  (deuxieme  vacciri}  which  has 
been  less  weakened.  The  animals  are  then  pro- 
tected against  the  most  virulent  anthrax,  but  only 
for  a  time.  From  such  a  culture,  however,  new 
cultures  of  virulent  bacilli  can  be  started,  and  a 
culture  that  is  "  vaccin  "  for  sheep  kills  a  guinea- 
pig,  and  then  yields  bacilli  that  are  fatal  to  sheep. f 
Exposure  to  a  temperature  of  55°  C.,  or  treatment 
with  *5  to  i  per  cent,  carbolic  acid,  deprives  the 

*  Pasteur,  Compt.  Rend.,  1861,  and  Revue  Scientifique,  1883. 
f  Klein,  Micro-organisms  and  Disease.     1885. 

19 


BACTERIOLOGY. 

bacilli  of  their  virulence.  The  virulence  of  the 
bacillus  is  also  altered  by  passing  the  bacillus 
through  different  species  of  animals.  The  bacillus 
of  sheep  or  cattle  is  fatal  when  re-inoculated  into 
sheep  or  cattle ;  but,  if  inoculated  in  mice,  the 
bacilli  then  obtained  lose  their  virulence  for  sheep 
or  cattle ;  only  a  transitory  illness  results,  and  the 
animals  are  protected  for  a  time  against  virulent 
anthrax.*  The  possibility  of  mitigating  the  virus 


FIG.  no. — SPORES  OF  BACILLUS  ANTHRACIS  UNSTAINED,  x  1500. 

depends  upon  the  species  of  animal ;  rodents  cannot 
be  rendered  immune  by  any  known  "  vaccin." 


METHODS     OF     STAINING    THE     BACILLUS    ANTHRACIS. 

Cover-glass  preparations  of  blood,  etc.,  can  be  stained 
with  a  watery  solution  of  any  of  the  aniline  dyes.  They 
may  be  rapidly  stained  with  a  drop  of  fuchsine  or  gentian 
violet  (p.  48),  but  more  satisfactorily  by  floating  the  cover- 
glasses  for  twenty-four  hours.  The  preparations  may  be 
dried  and  mounted  in  Canada  balsam,  but  the  typical 
appearances  are  best  observed  in  freshly-stained  specimens 
examined  in  water. 

The  sheath  and  protoplasmic  contents  can  be  differen- 
tiated by  staining  with  eosin  after  the  method  of  Gram. 

*  Klein,  Reports  of  the  Medical  Officer  of  the  Local  Government 
Board.  1882. 


SYSTEMATIC    AND    DESCRIPTIVE.  2QI 

The  spores  (Fig.  iio)are  not  stained  by  the  ordinary 
methods.  The  cover-glass  preparations  must  be  raised 
to  a  high  temperature  in  the  incubator,  or  treated  with 
sulphuric  acid  (p.  309),  or  passed  about  twelve  times 
through  the  flame  of  the  Bunsen  burner,  or  floated  on  hot 
solution  of  the  dye. 


FIG.  in. — SPORES  OF  BACILLUS  ANTHRACIS,  x  1200;  stained  with  gentian 
violet,  after  passing  the  cover-glass  twelve  times  through  the  flame. 

To  double-stain  spore-bearing  bacilli. — Float  the  cover- 
glasses  for  about  twenty  minutes  on  hot  alcoholic  solution 
of  fuchsine.  Decolorise  in  weak  hydrochloric  acid,  and 
after-stain  with  methylene  blue  (Fig.  112). 

Tissue  sections  are  best  stained  by  the  method  of  Gram, 


%   %  '     -;-  —-  :    -     1C-J 

•:'**'• 

FIG.  112.  -  FROM  A  DOUBLE-STAINED  PREPARATION  OF  BACILLUS 
ANTHRACIS,  x  1200. 

and  after-stained  with  eosin,  picrocarminate  of  ammonia, 
or  picro-lithium-carmine. 

A  more  rapid  double  stain  is  obtained  by  immersing  the 
sections  in  a  watery  solution  of  gentian-violet,  rinsing  in 
alcohol,  and  then  staining  by  the  method  of  Orth  (p.  61). 

Weigerfs  Method. — Place  the  sections  for  two  to  five 
minutes  in  a  I  per  cent,  watery  solution  of  gentian  violet 


BACTERIOLOGY. 


Wash  in  alcohol,  rinse  in  water,  and  transfer  to  picro- 
carmine  solution  (Weigert)  for  from  half  an  hour  to  an 
hour.  Treat  with  alcohol  till  the  colour  is  almost  washed 
out,  and  finally  clear  in  oil  of  cloves  and  mount  in  Canada 
balsam. 

Bacillus  mallei  (Bacillus  of  glanders).  —  Rods 
about  the  size  of  tubercle  bacilli  (Fig.  113).  When 
cultivated  on  solid  sterile  blood  serum  at  38°  C.,  the 
growth  appears  in  the  form  of  minute,  transparent 
drops  consisting  entirely  of  the  characteristic  bacilli. 
On  sterilised  potatoes  they  form,  in  a  week  to  ten 
days  at  37°  C.,  a  brown  gelatinous  layer.  Pure  cul- 


FIG.  113. — BACILLUS  MALLEI,  x  1200;  from  a  section  of  a  glanders' nodule. 

tivations  after  several  generations  produce  the  fol- 
lowing results  when  inoculated  into  horses,  rabbits, 
guinea-pigs,  and  field-mice.  A  spreading  ulcer  with 
indurated  base  appears  at  the  site  of  inoculation, 
while  smaller  ulcers  break  out  in  its  vicinity.  The 
lymphatics  become  swollen,  and  general  infection 
follows  in  the  form  of  nodules  in  the  internal  organs, 
and  nodules  and  ulcers  on  the  nasal  septum.  In 
guinea-pigs  a  characteristic  tumour  of  the  testis,  or 
ovary  and  vulva,  frequently  results,  and  should  be 
prepared  for  microscopical  sections.  The  bacilli 
are  found  in  the  nodules  of  the  nasal  mucous  mem- 


SYSTEMATIC    AND    DESCRIPTIVE.  2Q3 

brane,  the  lung,  spleen,  liver,  and  other  organs  in 
horses  and  sheep  affected  with  glanders. 

METHODS    OF    STAINING   THE    BACILLUS   OF   GLANDERS. 

The  bacilli  of  glanders  are  extremely  difficult  to  demon- 
strate. The  most  satisfactory  results  are  obtained  as 
follows  : — 

Method  of  Schutz. — The  sections  are  placed  for  twenty- 
four  hours  in  a  mixture  ot 

Potash  solution  (i  in  10,000);  {Equal 

Concentrated  alcoholic  methylene-blue  solution;)  parts. 

Wash  the  sections  in  a  watch-glass  with  water  acidulated 
with  four  drops  of  acetic  acid.  Transfer  for  five  minutes 
to  50  per  cent,  alcohol,  fifteen  minutes  to  absolute  alcohol, 
clarify  in  clove-oil,  and  mount  in  Canada  balsam. 

Bacillus  cedematis  maligni,  Koch  (Pas- 
tiur*  s  Septiccemia).  Rods  from  3 — 3*5  //,  long  and 
i — i 'i  /A  wide;  they  mostly  lie  in  pairs,  and  then 
appear  to  be  double  this  length.  The  rods  are 
rounded  at  their  ends,  and  form  threads  which  are 
sometimes  straight,  but  more  commonly  curved.  In 
stained  preparations  they  have  a  somewhat  granular 
appearance.  The  bacilli  are  distinguished  from 
anthrax  bacilli  by  their  being  somewhat  thinner, 
by  their  rounded  ends,  and  by  their  being  motile. 
Anthrax  bacilli  also  never  appear  as  threads  in  fresh 
blood,  and  are  differently  distributed  throughout 
the  body.  They  are  anaerobic,  and  can  be  culti- 
vated on  blood-serum  and  on  neutral  solution  of 
Liebig's  meat  extract  in  an  atmosphere  of  carbonic 


2 Q4  BACTERIOLOGY. 

acid.  By  embedding  material  containing  bacilli  in 
nutrient  agar-agar  and  nutrient  gelatine,  charac- 
teristic cultivations  are  obtained.  The  following 
process  may  be  adopted  to  obtain  a  pure  cultiva- 
tion.* A  mouse  inoculated  subcutaneously  with 
dust,  as  a  rule,  dies  in  one  to  two  days.  It  is  then 
pinned  out,  back  uppermost,  on  a  slab  of  wood 
(p.  113),  and  the  hair  singed  with  a  Paquelin's 
cautery  from  one  hind  leg  up  to  the  neck,  across 
the  latter,  and  down  again  to  the  opposite  hind 
leg.  Following  the  cauterised  line,  the  skin  is  cut 
through  with  sterilised  scissors,  and  the  flap  turned 
back  and  pinned  out  of  the  way.  With  curved 
scissors  little  pieces  of  the  subcutaneous  cedema- 
tous  tissue,  in  the  neighbourhood  of  the  inoculated 
spot,  are  cut  out,  and  sunk  with  a  platinum  needle 
in  a  i  per  cent,  nutrient  agar-agar,  or  5  per  cent, 
nutrient  gelatine.  Fragments  of  tissue  may  also 
be  embedded  by  the  method  already  described 

(P-  137). 

The  inoculated  tubes  are  placed  in  the  incubator. 
In  a  few  hours  a  whitish  turbidity  spreads  out  from 
the  piece  of  tissue,  and  upwards  in  the  needle  track. 
Examined  microscopically,  the  turbidity  is  found  to 
be  due  solely  to  the  development  of  bacilli  of  oedema. 
The  surface  exposed  to  the  air  exhibits  no  trace 
of  the  bacilli. 

To  investigate  the  tubes  microscopically,  a  steri- 
lised glass  tube  with  a  capillary  end  may  be  used, 

*  Hesse,  Deutsch.  Med.  Wocti.,No.  14.     1885. 


SYSTEMATIC    AND    DESCRIPTIVE.  295 

with  its  neck  plugged  with  sterilised  cotton  wool, 
and  provided  at  the  mouth  with  a  suction  ball. 
The  capillary  end  is  thrust  into  the  cultivation, 
and  a  small  fragment  removed  by  aspiration.  In 
the  course  of  the  first  day  the  bacilli  spread 
throughout  a  great  part  of  the  agar-agar  in  such 
a  way  that  a  more  or  less  equally  diffused  cloudi- 
ness of  the  medium  ensues,  with  subsequent  ap- 
pearance of  strongly  marked  clouds  or  lines  of 
turbidity.  At  the  same  time  gas  bubbles  develop 
along  the  needle  track,  and  a  collection  of  liquid 
takes  place,  while  spore-formation  also  commences. 
The  following  day  these  appearances  are  more 
marked,  the  opacity  is  more  pronounced,  the  develop- 
ment of  gas  increases,  and  the  liquid  contains  more 
spore-forming  bacilli  and  numerous  free  spores. 

The  nutrient  gelatine  cultures  during  the  first  day 
show  no  macroscopic  change,  but  after  a  few  days 
the  piece  of  tissue  is  surrounded  with  a  white  halo. 
This  gradually  spreads  in  all  directions,  and  is  ap- 
parently beset  with  hairs.  The  gelatine  liquefies, 
and  the  fragment  of  tissue,  degenerated  bacilli,  and 
spores,  sink  to  the  bottom.  The  cultivation  is  also 
very  characteristic  in  ^  per  cent,  nutrient  agar-agar. 
If  placed  in  the  incubator,  in  a  few  hours  a  cloudi- 
ness forms  around  the  piece  of  embedded  tissue, 
which  is  caused  by  bacilli  gradually  spreading  in 
all  directions  in  the  nutrient  medium.  Mice  inocu- 
lated from  these  cultivations  die  more  quickly  than 
from  the  original  infection  from  dust.  On  potatoes 


296  BACTERIOLOGY. 

they  are  cultivated  by  introducing  a  piece  of  liver  or 
other  tissue  containing  the  bacilli,  into  the  interior 
of  a  sterilised  potato  (p.  113),  incubated  at  38°  C. 
The  bacillus  is  not  deprived  of  its  virulence  by  cul- 
tivation. The  spores  of  the  cedema-bacilli  appear 
to  be  very  widely  distributed.  They  are  found  in 
the  upper  cultivated  layer  of  the  soil,  in  hay  dust, 
in  decomposing  liquids,  and  especially  in  the  bodies 
of  suffocated  animals,  which  are  left  to  decompose 
at  a  high  temperature.  From  any  of  these  sources 
animals  can  be  successfully  inoculated.  If  a  guinea- 
pig,  for  example,  be  subcutaneously  inoculated  with 
earth,  putrid  fluid,  or  hay  dust,  death  frequently 
occurs  in  from  twenty-four  to  forty-eight  hours.  At 
the  autopsy  the  most  characteristic  symptom  is  a 
widespread  subcutaneous  cedema,  which  originates 
from  the  point  of  inoculation,  accompanied  with  air- 
bubbles,  and  contains  a  clear  reddish  liquid  full  of 
motile  and  non-motile  bacilli.  The  internal  organs 
are  little  changed,  the  spleen  is  enlarged  and  of  a 
dark  colour,  and  the  lungs  are  hyperaemic,  and  have 
hsemorrhagic  spots.  Examined  immediately  after 
death,  few  or  no  bacilli  are  detected  in  the  blood  of  the 
heart,  but  in  that  of  the  spleen,  liver,  lungs, and  other 
organs,  in  the  peritoneal  exudation,  and  in  and  upon 
the  serous  coating  of  abdominal  organs  they  are 
present  in  large  numbers.  If,  on  the  other  hand, 
the  animal  is  not  examined  until  some  time  after 
death,  then  the  bacilli  are  found  in  the  blood  of  the 
heart,  and  distributed  all  over  the  body. 


SYSTEMATIC    AND    DESCRIPTIVE.  2Q7 

Bacillus  of  septicaemia  of  mice,  Koch. — 
Extremely  minute  bacilli,  *8 — i  p  long,  and  *i — '2 
broad,  often  in  pairs,  seldom  in  chai-ns  of  four..  On 
cultivation  they  do  not  appear  to  make  threads, 
but  the  bacilli  lie  together  in  masses.  Spores 
have  been  observed.  The  bacilli  are-  probably  non- 
motile.  They  are  most  commonly  in  the  interior 
of  white  blood  corpuscles.  In  these  they  increase, 
and  in  many  cases  a  white  cell  is  only  represented 
by  a  mass  of  bacilli.  The  bacilli,  or  rather  their 
spores,  occur  in  putrid  liquids.  If  a  number  of 
mice  are  inoculated  with  a  minimum  quantity  of 
putrid  fluid,  about  a  third  of  them  die  of  septi- 
caemia. They  rapidly  sicken,,  their  eyes  inflame, 
their  eyelids  stick  together,  they  become  soporific, 
and  die  in  from  about  forty  to  sixty  hours..  At  the 
autopsy  one  finds  slight  oedema  at  the  seat  of  inocu- 
lation, and  enlargement  of  the  spleen  ;  the  bacilli 
are  found  both  free  and  lodged  in  the  white  cor- 
puscles, in  the  cedematous  tissue,  and  in  the  blood 
capillaries.  A  minimal  quantity  of  this  blood 
produces  the  disease  if  inoculated  in  house-mice 
or  sparrows.  Field-mice  have  an  immunity.  Rab- 
bits and  guinea-pigs  inoculated  in  the  ear  suffer 
from  only  a  local  erythema,  which  disappears  after 
five  or  six  days,  and  renders  them  for  a  time  im- 
mune. Rabbits  inoculated  in  the  cornea  suffer 
from  an  intense  inflammation  of  the  eyes.  The 
bacilli  are  easily  cultivated  outside  the  body  on 
a  mixture  of  aqueous  humour  and  gelatine,  and 


298 


BACTERIOLOGY. 


especially  on  nutrient  gelatine  rendered  slightly 
alkaline  with  sodium  phosphate.  They  grow  also 
very  well  on  the  ordinary  nutrient  gelatine,  form- 
ing in  plate-cultivations  scarcely  perceptible  cloud- 
like  specks,  and  in  a  test-tube  of  nutrient  gelatine 
they  form  a  delicately  clouded  cultivation  along 

the  needle  track  (Figs. 
1 14  and  1 15).  A  small 
quantity  of  pure  culti- 
vation carried  through 
many  generations  re- 
produces the  disease 
when  inoculated  into 
mice.  The  organs 
should  be  hardened  in 
absolute  alcohol,  and 
sections  stained  prefer- 
ably by  the  method  of 
Gram  (Plate  XXII., 
Figs,  i  and  2). 

Bacillus  of  ulcer- 
ative  stomatitis   in 
the  calf,  Lingard  and 
Fig.  n4.  Fig.  nS.  Batt. — Rods  4  ju, — 8  /A, 

PURE-CULTIVATIONS  OF  THE  BACILLUS  •     i          .1    .    T 

OF  SEPTICAEMIA  OF  MICE  IN  NUTRIENT     or  more  in  lengtn  ,    I  /x, 

GELATINE-  in  width. 


Fig.  114.    In  two  days. 
Fig.  115.    In  five  days. 


Spores  are 

frequently  present.  In- 
jected into  the  rabbit  or  mouse  they  produce  a  fatal 
result.  They  were  observed  in  ulcerations  on  the 
tongue  and  mucous  membrane  of  the  mouth  of  calves. 


SYSTEMATIC    AND    DESCRIPTIVE.  2 99 

METHOD    OF    STAINING    THE    BACILLI    OF    ULCERATIVE 
STOMATATIS. 

Sections  through  the  ulcerations  of  the  calf  s  tongue,  or 
of  the  inoculated  tissue  of  the  rabbit,  were  stained  by 
immersion  in  a  mixture  of  magenta  and  methylene-blue. 
They  were  then  washed  in  spirit,  cleared  in  clove- oil,  and 
mounted  in  Canada  balsam. 

Bacillus  of  swine-typhoid  (Bacillus  of  swine- 
plague  or  swine-fever.  Bacillus  of  pneumo-enteritis  of 


FIG.  116. — FROM  A  PREPARATION  OF  BRONCHIAL  Mucus  OF  A  PIG 
[after  Klein]. 

the  pig,  Klein*). — Rods  2 — 3  p,  long,  actively 
motile  (Fig.  116);  spore -formation  described  (Fig. 
118).  They  can  be  cultivated  in  broth  and  hydro- 
cele-fluid,  and  carried  on  through  successive 
generations.  A  drop  of  any  of  these  cultivations 
produces  the  disease  in  pigs,  mice,  and  rabbits 
(Fig.  117);  the  animals  die  with  a  characteristic 
swelling  of  the  spleen,  coagulative  necrosis  of 
tracts  of  the  liver  tissue,  and  inflammation  of  the 

*  Klein,  Report  to  Med.  Offic.  Loc.  Govt.  Board.     1877—1878, 


3OO  BACTERIOLOGY. 

lungs  ;  pigs  inoculated  with  artificial  cultures  are 
protected  against  a  fatal  attack. 

The  bacillus  was  observed  in  the  diseased  organs 


9 

FIG.  117. — BLOOD  OF  FRESH  SPLEEN  OF  A  MOUSE,  AFTER  INOCULATION 
WITH  SWINE  FEVER  [after  Klein]. 

of  pigs  that  had  died  of  swine  fever,  and  of  animals 
that  had  died  from  the  inoculated  disease. 


»-l, 


FIG.  1 18.— BACILLI  FROM  AN  ARTIFICIAL  CULTURE,  WITH  SPORES 
[after  Klein], 

Bacillus  of  swine-erysipelas  (Bacillus  cies 
erysipelas  malignum,  of  Rothlauf,  or  Rouget  du  pore). 
Extremely  minute  bacilli,,  bearing  a  close  re- 
semblance to  the  bacilli  of  septicaemia  of  mice.* 
In  test-tubes  of  nutrient  gelatine  they  develop  a 
cloudy  growth  in  the  needle  track  (Plate  XXVI., 

*  Loffler  und  Schiitz,  Arbeit.en  aus  dem  Kaiser  lichen  Gesu?id- 
heitsamte,.  vol.  i.  1885. 


SYSTEMATIC    AND    DESCRIPTIVE.  30! 

Fig.  i),  and  in  plate-cultivations  characteristic, 
thread-like,  branching,  or  star-like  colonies  are 
formed  (Plate  XXVI.,  Fig.  2).  Inoculated  into 
mice  and  rabbits,  a  fatal  result  is  produced  ;  but 
experiments  with  pigs  were  unsuccessful.  Pigeons 
were  also  susceptible,  and  the  bacilli  were  detected 
in  their  blood  (Plate  XXVI.,  Fig.  3). 

Bacillus  in  tetanus,  Nicolaier. — Rods,  some- 
what longer  but  scarcely  thicker  than  the  bacillus 
of  mouse-septicaemia.  Occasionally  thread-forms 
result,  but  they  are  collected  mostly  in  irregular 
masses.  They  exhibit  a  characteristic  spore-for- 
mation. They  were  found  associated  with  other 
bacteria  in  abscesses  resulting  from  the  inoculation 
of  mice  and  rabbits  with  garden  earth.  Inoculation 
of  earth  subcutaneously  in  these  animals  induces 
fatal  tetanus.  A  cultivation  of  the  mixture  of 
micro-organisms  on  blood  serum  also  produced 
the  same  disease.  Bacilli  stated  to  be  identical 
with  the  bacilli  of  earth-tetanus  have  been  ob- 
served in  a  case  of  tetanus  in  man.  Further 
researches  are  required  to  establish  their  patho- 
genic properties. 

Bacillus  alvei,  Cheshire  and  Cheyne.* — Rods 
varying  in  size,  and  forming  large  oval  spores. 
When  cultivated  in  nutrient  gelatine  in  test-tubes 
a  delicate,  ramifying  growth  appears  on  the  sur- 
face, and  irregular  whitish  masses  arise  along  the 

*  Cheshire  and   Cheyne,  Journ.   Royal  Microscopical    Society, 
1885,  pp.  582-601. 


302  BACTERIOLOGY. 

needle  track.  Processes  shoot  out  from  these 
masses,  and  extend  through  the  gelatine  for  long 
distances.  They  are  thickened  at  points  in  their 
course,  and  clubbed  at  the  ends.  The  gelatine  is 
gradually  liquefied,  and  the  bacilli  form  a  loose, 
white,  flocculent  deposit  at  the  bottom  of  the  tube. 
The  liquid  in  the  tube  becomes  yellowish  in  colour 
after  a  time,  and  gives  off  an  odour  of  stale  but 
not  ammoniacal  urine.  The  colour  and  odour  are 
distinctive  also  of  the  disease  attributed  to  the 
bacilli.  In  plate-cultivations,  the  bacilli  grow  out 
in  series  of  rods  in  single  file,  or  in  rows  of  several 
side  by  side.  The  processes  which  are  formed, 
tend  to  .curve,  and  at  a  short  distance  from  the 
track  of  the  needle-streak  form  a  distinct  circle, 
from  which  another  process  grows  out,  and  a  fresh 
circle  is  developed.  The  gelatine  in  the  vicinity 
of  the  bacilli  gradually  liquefies,  and  channels  are 
formed  in  the  gelatine  in  which  the  bacilli  move 
backwards  and  forwards.  On  nutrient  agar-agar 
a  whitish  layer  develops,  consisting  of  bacilli 
arranged  side  by  side,  which  in  a  few  days  are 
replaced  by  rows  of  spores  similarly  arranged.  On 
potatoes  they  form  a  dryish  yellow  layer,  and  in 
milk  a  tremulous  jelly.  A  cultivation  of  the  bacillus 
in  milk,  sprayed  over  a  honeycomb  containing  a 
healthy  brood  of  bee  larvae,  produced  the  disease 
known  as  "  foul  -brood."  Adult  bees  fed  on  ma- 
terial containing  bacilli  became  affected ;  inocula- 
tion of  mice  and  rabbits  with  the  bacillus  gave 


SYSTEMATIC    AND    DESCRIPTIVE.  303 

doubtful  results.  The  bacilli  were  isolated  from 
the  diseased  larvae  of  bees. 

Bacillus  pyocyaneus  (Micrococcus  pyocyanem, 
Gessard.  Bacterium  czruginoMim,  Schroter.  Ba- 
cillus fluorescens). — Slender  rods  sometimes  linked 
in  twos  or  threes,  or  collected  in  irregular  masses. 
Spore-formation  present.  On  plate-cultivations 
white  colonies  with  indistinct  contour  appear  in 
twenty-four  hours,  and  the  whole  of  the  gelatine 
has  a  greenish  shimmer.  In  test-tube  cultivations 
the  gelatine  is  liquefied,  and  coloured  green  by 
reflected  light,  and  a  deep  orange  by  transmitted 
light  (Plate  V.,  Fig.  i).  On  nutrient  agar-agar 
they  form  a  white  layer,  and  colour  the  medium 
a  pea-green.  The  pigment  formed  by  the  rods  is 
a  definite  principle,  pyocyanin.*  It  can  be  ex- 
tracted with  chloroform  from  pus,  and  from  washings 
of  bandages ;  it  is  soluble  in  acidulated  water,  which 
it  colours  red.  In  neutral  solution  it  becomes  blue. 
It  crystallises  in  chloroform  in  long  needles ;  and 
forms  sometimes  lamellae  and  prisms.  The  rods 
occur  in  the  pus  of  those  cases  in  which  the  wounds 
and  pus-stained  bandages  exhibit  a  greenish-blue 
colour. 

Bacillus  ianthinus  {Bacterium  ianthinum,  Zopf. 
Bacillus  violaceus). — Slender  rods,  about  four  times 
their  width  in  length,  with  rounded  ends.  They 
also  form  threads,  and  are  actively  motile.  Spore- 

*  Gessard,  De  la  Pyocyanine  et  de  Son  Microbe.     Paris  these. 
1882. 


304  BACTERIOLOGY. 

formation  present  in  the  rods.  On  plate  cultiva- 
tions the  colonies  occur  as  circumscribed  liquefied 
areas,  in  the  centre  of  which  is  a  collection  of  the 
coloured  growth.  In  test-tubes  a  funnel-shaped 
liquefaction  takes  place,  while  a  granular-looking 
violet  mass  subsides  to  the  bottom.  On  agar-agar 
and  potatoes  a  beautiful  violet  growth  rapidly 
develops.  They  were  observed  on  pieces  of  pigs' 
bladder  floating  on  the  surface  of  water  rich  in 
bacteria.  They  occurred  only  on  the  surface  of 
the  bladder  exposed  to  the  air,  and  never  on  the 
part  under  water.  They  occasionally  occur  in 
common  tap  water.  The  colouring  matter  is 
soluble  in  alcohol. 

Bacillus  cyanogenus,  Fuchs.  (Bacterium 
syncyanum.  Bacillus  of  Blue  Milk). — Motile  rods, 
2 '5 — 3*5  p,  in  length,  and  "4  //,  wide  (Fig.  119). 
The  rods  after  division  may  remain  linked 
together,  and  form  chains.  Non- motile  rods 
enveloped  in  a  gelatinous  capsule,  and  involution- 
forms,  have  also  been  described.  Spore-formation 
present. 

Cultivated  in  a  test-tube  of  nutrient  gelatine,  the 
bacilli  grow  principally  upon  the  free  surface,  in 
the  form  of  a  white  layer.  The  surface  of  the 
gelatine  becomes  cupped,  and  a  peculiar  greenish - 
brown  colour  develops  in  the  medium,  especially 
in  proximity  to  the  growth. 

On  a  sloping  surface  of  nutrient  agar-agar, 
they  grow  as  a  white  layer,  and  colour  the. 


SYSTEMATIC    AND    DESCRIPTIVE. 


305 


upper  part  of  the  medium  a  smoky  brown  (Plate 
II.,  Fig.  2). 

The  bacilli  can  also  be  cultivated  in  milk  and  on 
various  other  substrata,  as  potatoes  (Plate  XXL), 
boiled  rice,  and  starch.  A  pure  cultivation  in 
neutralised  sterile  milk  develops  a  weak  alkaline 


E 

0 

c 

it 

/\] 

('' 

>   n 

.' 

\\ 

',</ 


FIG.  119. — BACILLUS  CYANOGENUS,  x  650. 

A.  Active  rods.  B.  Rods  in  zooglcea.  C.  Chain  of  short  rods.  D.  Chain  of 
cocci.  E.  Cocci  stage.  F,  G.  Spore-forming  rods.  H.  Involution-forms 
[  After  Neelsen]. 

reaction.  The  colouring  matter  which  is  formed, 
varies  with  the  nourishing  medium  ;  for  example, 
in  milk  a  slate-blue  coloration  is  produced,  but 
if  the  milk  has  become  acid  by  the  growth  of 
the  Bacillus  acidi  lactici,  then  the  colour  is  an 
intense  blue 

The    micro-organism    occurs     occasionally     in 

20 


306  BACTERIOLOGY. 

cow's  milk,  producing  a  blue  colour.  It  has  been 
observed,  especially  in  the  north  of  Germany, 
during  the  warm  months ;  and  where  milk  is 
kept  in  hot  rooms,  in  the  winter  also.  The  blueing 
was  originally  attributed  to  a  diseased  condition 
of  the  cows,  or  to  their  eating  certain  meadow 
plants. 

Bacillus  acidi  lactici. — Long  and  short  rods, 
i — 2'8ft  long,  *3 — '4  p,  thick,  and  thread-forms;  no 
cocci ;  spore-formation.*  Cultivated  on  nutrient 
gelatine  the  breadth  of  the  rods  is  lessened.  They 
grow  best  between  35  and  42°  C.,  and  cease  under 
io°C.  Cultivated  at  a  temperature  over  45*5°,  they 
are  no  longer  able  to  produce  acidity.  Probably 
several  micro-organisms  are  able  to  produce  an 
acid  reaction  in  milk. 

They  occur  with  various  other  bacteria  in  sour 
milk,  and  a  pure  cultivation,  isolated  by  plate- 
cultivations,  turns  sterilised  milk  sour. 

Bacillus  Fitzianus,  Zopf. — Cocci,  short  rods, 
long  rods,  and  threads.  This  bacillus,  cultivated 
in  meat  extract  and  glycerine  at  36°  C.,  causes  an 
active  fermentation  with  the  production  of  ethyl 
alcohol.  Spore-formation  occurs  in  the  rods. 
Observed  in  unboiled  hay  infusion,  accompanying 
the  hay  bacillus. 

Bacillus  subtilis  (Hay  bacillus). — Cylindrical 
rods  as  much  as  6  ft  in  length,  and  about  three 
times  as  long  as  broad.  Single  forms  grow  to 

*  Hueppe,  Mittheil  a.d.  Gesundheitsamt,  N.  Band. 


SYSTEMATIC    AND    DESCRIPTIVE.  307 

double  their   length,   and   then    undergo    division.. 
They  also   form  threads  which   may  be  composed 
of  long   rods,    short  rods,    and  cocci.      They   are 
motile,  and  provided  with  a  flagellum  at  each  end. 
If    the    nourishing    medium    is    impoverished,    the 
multiplication   of   the    rods    by    division    gradually 
ceases,  and  spore-formation  commences.     The  rods 
become  motionless,  and  a  dark  spot  is  visible,  either 
in  the  middle  or  towards  one  end.     This  gradually 
develops  into  a  shining  spore  with  a  dark  contour. 
The  rods  swell  slightly  during  this  process,   their 
contour   becomes   undefined,  and  soon  disappears 
entirely,  so  that  the   spores  are  set  free  in  about 
twenty-four  hours.     The  spores  are  1*2  //,  long,  and 
•6  p,  broad.    They  develop  into  rods  in  the  following 
way.     On  one  side  of  the  spore  a  swelling  appears, 
at  the  summit  of  which  an  opening  in  the  spore- 
membrane  results,  and  the  germ    escapes.      This 
lengthens    into    a    rod,    and    remains    for   a   time 
attached    to   the   empty   spore-membrane.      These 
spores  are  widely  distributed,  and  occur  in  the  air, 
soil,  dust,  etc.     On   the  excrement  of  herbivorous 
animals  the  bacilli  form  a  white  efflorescence,  and 
on  infusion  of  horse-dung  a  thick  crumpled   skin. 
They   flourish   equally  in  liquids  and  upon   damp, 
solid,  nourishing  media.     On  potatoes  they  grow  as 
a  yellowish-white  skin  ;  on  ordinary  nutrient  liquids 
they    develop   a   thin,    and   subsequently  a    thick, 
dense,  crumpled  pellicle,  with  copious  spore-forma- 
tion.    They   are   aerobic;    deprivation    of    oxygen 


308  BACTERIOLOGY. 

causes  the  growth  of  the  bacilli  to  cease,  and  the 
rods  degenerate.  They  may  be  cultivated  in 
various  other  nourishing  media,  such  as  blood- 
serum,  nutrient  gelatine,  and  nutrient  agar-agar 
(Plate  XIV.,  Fig.  2). 

The  simplest  way  to  obtain  a  culture  of  the 
bacillus  is  to  make  a  decoction  of  hay.  The  hay 
is  chopped  into  small  pieces,  and  boiled  with  dis- 
tilled water  in  a  flask  for  a  quarter  of  an  hour ;  it  is 
then  filtered  into  a  beaker,  which  must  be  covered 
with  a  glass  plate,  and  set  aside  in  a  warm  place. 
In  two  or  three  days  the  liquid  swarms  with  the 
bacilli,  the  spores  of  which  exist  in  great  numbers 
in  ordinary  hay.  A  more  sure  method  for  obtaining 
a  pure  cultivation  is  as  follows : — 

(a)  Add  only  a  small  quantity  of  water  to  some 
finely  chopped  hay,   and  set  aside  for  four  hours 
at  36°  C. 

(b)  Pour   off   the  extract,   and   dilute  it   to    the 
Sp.  Gr.  1-004. 

(c)  Boil  gently  for  one  hour  in  a  bulb  plugged 
with  cotton  wool. 

(d)  Set  aside  500  ccm.  of  the  extract  at  36°  C. 
In  about  twenty- four  hours,  as  a  rule,  a  pellicle 

has  commenced  to  develop  upon  the  surface  of 
the  liquid.  If  the  reaction  is  definitely  acid, 
carbonate  of  soda  solution  must  be  added  to  the 
decoction. 


SYSTEMATIC    AND    DESCRIPTIVE.  309 

METHODS    OF    STAINING    HAY    BACILLUS. 

To  demonstrate  the  flagella  of  the  bacilli,  they  may  be 
stained  with  haematoxylin  solution  (Koch). 

The  linking  together  of  cocci,  long  rods,  and  short  rods 
in  the  threads,  is  shown  by  treating  with  alcoholic  solution 
of  fuchsine,  or  with  iodine  solution  (Zopf). 

To  stain  the  spores  the  cover-glass  preparations  must  be 
heated  to  a  very  high  temperature  (210°  C.),  in  the  hot-air 
steriliser  for  half  an  hour,  or  they  may  be  exposed  for  a 
few  seconds  to  the  action  of  concentrated  sulphuric  acid 
(Biichner),  or  floated  for  twenty  minutes  on  hot  solution  of 
the  dye. 

Bacillus  ulna,  Cohn. — Cocci,  short  rods,  long 
rods,  and  threads.  Diam.  of  the  cocci  1*5 — 2*2  p,. 
Spore-formation  in  both  short  and  long  rods.  No 
septic  odour  is  produced  by  this  bacillus  in  a 
nourishing  liquid.  Cloudy  masses  are  found  on  the 
surface  of  the  liquid,  which  later  form  a  thick  dry 
pellicle.  The  latter  consists  of  bundles  of  threads 
matted  together.  The  formation  of  ellipsoidal 
spores  occurs  in  the  usual  way ;  they  measure 
2*5 — 2*8  ft  long,  and  more  than  i  ^  wide.  The 
bacillus  is  found  in  rotting  eggs,  and  can  be  culti- 
vated on  boiled  white  of  egg.  It  is  closely  allied 
to  Bacillus  subtilis. 

Bacillus  tumescens,  Zopf.* — Cocci,  long  and 
short  rods.  They  form  a  jelly-like  disc,  "5 — i  cm. 
in  diam.,  on  slices  of  boiled  carrot,  with  the  appear- 
ance of  a  rather  tough  crumpled  skin  of  a  whitish 
colour.  Examination  of  this  pellicle  shows  that  it 

*  Zopf,  Die  Spaltpilze.     1885. 


3io 


BACTERIOLOGY. 


is  formed  of  rows  of  rods  lying  closely  together. 
These  rods  can  be  observed  to  divide  into  short 
rods  and  cocci.  Spore-formation  occurs  in  two 
stages  of  development,  viz.,  in  the  cocci  and  in  the 
'short  rods.  A  cultivation  is  obtained  by  exposing 
slices  of  boiled  carrot,  slightly  moistened,  to  the 
air  at  the  temperature  of  the  room. 

Bacillus  megaterium,  De  Bary. — Large  rods 
2 '5  n  wide,  and  four  to  six  times  as  long.     They 


FIG.  120.— BACILLUS  MEGATERTUM. 

a.  A  chain  of  rods,  X  250.     The  rest  X  600. 

b.  Two  active  rods. 

dtof.  Successive  stages  of  germination. 
h  and  l»  Successive  stages  of  germination. 
[After  De  Bary.] 

are  usually  somewhat  curved.  Transverse  division 
•occurs,  each  segment  attaining  the  length  of  the 
original  rod.  In  the  fresh  state  they  appear  non-arti- 
culated, but  when  treated  with  a  dehydrating  agent 
.(tincture  of  iodine,  alcohol),  they  are  seen  to  be 
composed  of  short  segments.  The  rods  are  motile,, 
and  form  irregular  chains,  of  a  disjointed  appear- 


SYSTEMATIC    AND    DESCRIPTIVE. 


ance.  They  can  be  cultivated  on  nutrient  agar- 
agar  and  nutrient  gelatine.  The  latter  is  slowly 
liquefied,  but  the  appearances  are  not  characteristic. 
Spore-formation  occurs  in  the  usual  jjjp 
way  (Fig.  120).  It  was  first  observed 
on  boiled  cabbage. 

Bacillus  figurans  (Wurzel  Ba- 
cillus ,  Bacillus  my  co  ides,  Fliigge).  — 
Rods,  with  rounded  ends,  varying  in 
length.  Spore-formation  present.  In 
plate-cultivations  they  cause  a  cloudy 
growth,  spreading  from  various  points; 
if  a  cover-glass  impression  is  made, 
this  is  found  to  consist  of  the  regularly- 
arranged  parallel  rods.  The  chains  of 
rods  become  twisted  at  intervals  into 
curious  convolutions,  from  which  off- 
shoots are  continued  in  various  direc- 
tions. These  long  shoots  or  processes 
are  again  twisted  at  intervals  into 
varying  shapes  and  patterns  (Plate 
XXV.,  Figs,  i  and  2).  Cultivated  in 
nutrient  gelatine,  the  bacilli  form  on 
the  surface  visible  windings,  from 
which  fine  filaments  grow  down  into  FlG  I2I 

the  gelatine.     They  spread  out  also  in  PURE  CULTIVA- 

TION OF  BACII.- 


almost  parallel  lines  transversely  from  LUS 

IN     NUTRIENT 

the  needle  track.     On  an  oblique  sur-  AGAR-AGAR. 
face   of  nutrient   agar-agar   the    filaments    spread 
downwards    into    the    substance   of  the  jelly,  and 


3  I  2  BACTERIOLOGY. 

outwards  from  the  central  streak  on  the  surface, 
forming  a  feather-like  cultivation*  (Fig.  121).  They 
are  present  in  garden  earth,  and  have  also  been 
cultivated  from  the  air  when  charged  with  dust 
raised  from  the  soil. 

Bacillus  tremulus. — Rods  shorter  and  thinner 
than  those  of  Bacillus  subtilis.  They  are  provided 
with  a  flagellum  at  both  ends,  and  exhibit  charac- 
teristic trembling  and  rotatory  movements.  Spores 
thicker  than  the  bacillus,  and  often  placed  laterally. 
They  were  observed  on  rotting  plant  infusions, 
forming  a  thick  slimy  skin. 

Bacillus  of  jequirity,  Sattler. — Rods  2 — 4-5  p, 
long  and  "58  ^  thick.  They  can  be  cultivated  on 
nutrient  gelatine  and  blood  serum.  Infusion  of  je- 
quirity containing  the  bacilli,  or  an  artificial  cultiva- 
tion of  the  bacilli,  inoculated  into  the  conjunctiva 
of  healthy  rabbits  produces  severe  ophthalmia.  The 
poisonous  principle  is,  however,  believed  to  be  a 
chemical  ferment,  abrin,z.r\&  not  the  bacillus.  Boiling, 
which  does  not  destroy  the  spores  of  the  bacillus, 
destroys  the  ferment,  and  cultivations  started  with 
these  spores,  though  teeming  with  jequirity  bacilli, 
are  quite  harmless.!  The  bacilli  occur  in  infusions 
of  the  beans  of  Abrus  precatorius,  or  jequirity. 

Bacillus  caucasicus,  Kern. — Rods  forming 
two  spores,  one  at  each  end,  otherwise  similar  to 


*  Described  by  the  author,  "  Notes  from  a  Bacteriol.  Laboratory," 
Lancet.     1885. 

t  Klein,  Micro-organisms  and  Disease.    1885. 


SYSTEMATIC    AND    DESCRIPTIVE.  313 

Bacillus  subtilis.  They  occur  in  the  form  of  whitish 
lumps  in  company  with  Saccharomyces  mycoderma 
in  the  production  of  a  drink  "  kephir  "  from 
cow's  milk.  The  fermentation  is  not  due  to  the 
bacillus. 

Bacillus  dysodes,  Zopf. — Cocci,  long  and  short 
rods,  and  spores.  They  were  observed  in  bread, 
making  it  greasy  and  unfit  for  food,  and  generating 
a  penetrating  odour  resembling  a  mixture  of  pep- 
permint and  turpentine.  A  great  loss  may  result 
to  bakers  if  the  fungus  is  introduced  with  the 
yeast. 

Bacillus  Hansenii,  Rasmussen. — Rods  2-8 — 6 
JJL  long,  '6 — '8  ^  wide.  Cultivated  on  sterilised  potato 
in  four  days  they  form  a  chrome-yellow  layer  with 
an  agreeable  fruitlike  smell.  Two  or  three  days 
later  the  growth  dries,  and  changes  to  orange-yellow 
in  colour ;  later  it  passes  to  yellowish  or  brown, 
and  forms  at  the  same  time  spores  1*7  /x,  long,  IT 
/x,  wide.  The  colouring  matter  is  insoluble  in  most 
reagents. 

The  bacilli  occur  on  nourishing  solutions,  malt 
infusion,  broth,  wine,  which  have  been  kept  at  31 
to  33°  C.,  as  a  yellow  or  whitish  skin. 

Bacillus  erythrosporus,  Cohn. — Motile  rods 
and  threads ;  rods  exhibiting  spore- formation. 
They  grow  well  in  nutrient  gelatine,  colouring  the 
medium  green  by  transmitted  light.  They  were 
found  to  form  a  pellicle  on  meat-extract-solutions 
and  on  rotting  albuminous  liquids. 


314  BACTERIOLOGY. 

Bacillus  septicus,  Klein.*— Rods  varying  in 
size,  non-motile.  They  form  threads  or  leptothrix 
filaments,  and  are  rounded  at  the  ends.  They  are 
anaerobic,  and  form  spores  independently  of  access 
of  air.  In  a  nourishing  fluid  they  are  overcome 
by  the  presence  of  micrococci,  Bacterium  termo  or 
Bacillus  subtilis.  They  occur  in  the  soil,  in  putrid 
blood,  and  many  putrid  albuminous  fluids,  and 
occasionally  in  the  blood-vessels  of  man  and 
animals  after  death. 

Bacillus  saprogenes,  Rosenbach. — Three  rod- 
formed  organisms  have  been  described  by  Rosen- 


FIG.  122. — BACILLUS  SAPROGENES,  No.  i.     [After  Rosenbach.] 

bach  as  intimately  associated  with  putrefactive 
processes. 

No.  i. — Large  rods  (Fig,  122),  which  form  an 
irregular  sinuous  streak  with  a  mucilaginous  ap- 
pearance, when  cultivated  on  nutrient  agar-agar. 
Spore-formation  present.  They  grow  also  very 
readily  on  blood  serum,  and  all  cultivations  yield 
the  odour  of  rotting  kitchen  refuse.  They  are  not 
pathogenic. 

No.  2. — Rods  shorter  and  thinner  than  No.  i. 
They  develop  very  rapidly  on  agar-agar,  forming 

*  Klein,  Micro-organisms  and  Disease.     1885. 


SYSTEMATIC    AND    DESCRIPTIVE.  315 

transparent  drops,  which  become  grey.  They  were 
isolated  from  a  patient  suffering  from  profusely- 
sweating  feet.  The  cultivations  yield  a  character- 
istic odour  similar  to  the  last.  They  are  pathogenic 
in  rabbits.  They  appear  to  be  identical  with 
Bacillus  foetidus  (Bacterium  foetidum.  Thin). 

No.  3. — See  Bacterium  saprogenes. 

Bacillus  foetidus  (Bacterium  foetidum,  Thin). — 
Cocci,  short  rods,  long  rods,  and  leptothrix.  The 
cocci,  1*25 — i '4  in  diam.,  occur  singly  or  in 
pairs.  Spore-formation  present  in  the  rods.  They 
were  isolated  from  the  exudation  in  a  case  of  profuse 
sweating  of  the  feet,  and  the  odour  was  noticeable 
in  the  cultivation  (vide  Bacillus  saprogenes). 

Bacillus  putrificus  coli,  Bienstock. — Slender, 
motile  rods,  3  p,  in  length,  often  less,  sometimes 
forming  long  threads.  Spore-formation  present. 
Cultivations  in  gelatine  are  iridescent.  They  are 
constantly  present  in  faeces. 

Bacillus  saprogenes  foetidus,  Schottelius. — 
Rods  with  rounded  ends,  shorter,  but  about  same 
width  as  the  hay- bacillus.  Immotile;  spore-forma- 
tion present.  On  nutrient  gelatine  the  colonies  are 
yellowish,  and  do  not  liquefy  the  medium.  On 
potatoes  they  form  a  pale  grey  layer.  They  develop 
a  strong  rotting  odour.  They  were  isolated  from 
the  organs  and  intestinal  contents  of  pigs  reputed 
to  be  ill  with  swine-erysipelas. 

Bacillus  aerophilus,  Liborius. — Slender  rods, 
two-thirds  the  width  of  the  hay-bacillus,  and 


3  1 6  BACTERIOLOGY. 

thread-forms.  Spore-formation  present.  In  nutrient 
gelatine  they  form  dot-like  colonies  of  greenish- 
yellow  colour,  which  liquefy  the  gelatine.  In  test- 
tubes  a  somewhat  funnel-shaped  liquefaction  results. 
On  potatoes  they  develop  a  yellowish  layer.  Power- 
fully aerobic.  Found  as  a  contamination. 

Bacillus  mesentericus  fuscus,  Flugge. — 
Small,  short,  actively-motile  bacilli,  often  linked  in 
twos  and  fours.  Spore-formation  present.  They 
form  white  colonies  on  plate-cultivation,  which  later 
stream  out  in  rays  at  the  periphery,  and  liquefy  the 
gelatine.  In  test-tube  cultivations  a  funnel-shaped 
turbidity  is  produced,  and  then  a  stratum  of 
liquefied  gelatine  with  subsiding  flocculi.  On 
potatoes  they  develop  a  smooth  yellowish  layer, 
which  soon  becomes  folded  and  wrinkled,  forming 
a  delicate  veil  over  the  nutrient  surface.  They 
occur  on  unsterilised  potatoes. 

Bacillus  mesentericus  vulgatus,  Flugge 
(Potato  bacillus). — Rods,  longer  and  thicker  than  the 
above,  and  sometimes  thread-forms;  spore-for- 
mation present.  The  colonies,  at  first  somewhat 
transparent,  have  later  an  opaque  centre,  and 
liquefy  the  gelatine.  In  test-tubes  of  nutrient 
gelatine  a  funnel-shaped  turbidity  results,  and 
then  an  upper-stratum  is  completely  liquefied, 
while  a  skin  floats  on  the  surface,  and  flocculent 
masses  subside  to  the  bottom  of  the  liquid  layer. 
They  occur  on  potatoes. 


SYSTEMATIC    AND    DESCRIPTIVE, 


317 


Genus  V. —  Vibrio. 
SPECIES. 

UNASSOCIATED  WITH  DISEASE  : — 

Vibrio  rugula      ...         .     Zymogenic  saprophyte. 

Vibrio  rugula,  Miiller.— Rods  and  threads, 
6 — 16  ft  long,  about  '5 — 2*5  thick.  The  rods  are 
either  simply  bowed,  or  possessed  of  one  shallow 
spiral  (Fig.  123).  They  bear  a  flagellum  at  each 


FIG.  123.— VIBRIO  RUGULA,  x  1020.  A.  Bowed  threads.  B.  Slightly-curved 
rods.  C.  Rods  swollen  preparatory  to  spore-formation.  D.  Rods  swollen 
at  the  spore-forming  end.  E.  Various  stages  of  the  developing  spores. 
[After  Prazmowski.j 

end.  The  rods  form  swarms  when  causing  de- 
composition, and  then,  or  after,  grow  out  into 
threads,  curved  in  a  screw-like  manner.  In  the 
next  stage  of  development  the  rods  cease  to  move; 
and  become  swollen  with  granular  contents.  One 


3  I  8  BACTERIOLOGY. 

extremity  develops  an  enlargement,  giving  the  rod 
the  appearance  of  a  pin.  The  spore  formed  by  the 
contraction  of  the  plasma  in  the  swollen  end  finally 
becomes  globular.  The  vibrios  appear  in  vegetable 
infusions,  causing  fermentation  of  cellulose. 


Genus  VI. — Clostridium. 
SPECIES. 

ASSOCIATED  WITH  DISEASE  IN  ANIMALS  :— 

Clostridium  of  symptomatic  anthrax     Pathogenic. 

UNASSOCIATED  WITH  DISEASE  : — 

Clostridium  butyricum     .         .         .     Zymogenic  saprophytes. 
Clostridium  polymyxa      ...  ,,  ,, 

Clostridium  butyricum,  Prazmowski  (Bacillus 
amylobacter^  Van  Tieghem  ;  Bacillus  butyricus. 
Bacillus  of  butyric  acid  fermentation}. — Rods  3  — 10 
p,  long,  and  under  i  ft  wide,  often  indistinguish- 
able from  Bacillus  subtilis.  They  grow  out  into 
long,  apparently  unjointed  threads.  They  are 
mostly  actively  motile,  but  also  occur  in  zooglcea. 
The  rods  and  threads  are  sometimes  slightly  bent 
like  vibrios.  They  are  anaerobic.  The  shorter  rods 
as  a  rule  swell  in  the  middle,  becoming  ellipsoidal, 
lemon  or  spindle-shaped  ;  the  long  rods,  and  some- 
times the  short  ones,  swell  at  one  end ;  in  either 
case  ellipsoidal  spores  are  developed  (Fig.  124). 

If  they  be  cultivated  in  nutrient  gelatine,  the 
medium  is  liquefied,  and  a  scum  formed  on  the 
surface.  They  grow  best  between  35°  and  40°  C. 
The  spores  are  widely  distributed  in  nature,  and 


SYSTEMATIC    AND    DESCRIPTIVE. 


319 


grow  readily  on  fleshy  roots,  old  cheese,  etc.  They 
convert  the  lactic  acid  in  milk  into  butyric  acid, 
and  produce  the  ripening  of  cheese.  They  occur 


FIG.  124.— CLOSTRIDIUM 

A.  Active  stage,     (a,  b]  Bent  rods  (vibrio-form)  and  threads.     (_c}  Short  rods. 

(//)  Long  rods. 

B.  Spore-formation.     C.  Spore*germination.     [After  Prazmowski.] 

also  in  solutions  of  starch,  dextrine,  and  sugar, 
and  are  the  active  agents  in  the  fermentation  of 
sauerkraut  and  sour  gherkins. 


32O  BACTERIOLOGY. 


METHOD    OF    STAINING    THE    BACILLUS    OF    BUTYRIC 
ACID    FERMENTATION. 

Treat  the  bacilli  with  iodine-solution.  At  certain  stages 
of  the  fermentation-process  the  plasma  takes  a  blue  or 
violet-black  coloration.  The  young  rods  give  the  former 
appearance,  and  the  older  ones  the  latter.  It  is  most 
easily  observed  when  the  bacillus  is  cultivated  in  a  sub- 
stance containing  starch,  or,  if  starch  is  wanting,  in  the 
presence  of  cellulose,  calcium-lactate,  or  glycerine;  in 
bacilli  cultivated  in  sugar  solutions  the  reaction  seldom 
appears. 

Clostridium  polymyxa,  Prazmowski. — Threads 
consisting  of  rods  which  vary  in  length  ;  cocci, 
involution-forms,  and  spores  are  also  present ;  cul- 
tivated on  nourishing  solutions  they  develop  a  thick 
skin  on  the  surface.  On  boiled  beet  and  other 
roots  they  form  a  gelatinous  scum,  which  often 
consists  of  crinkled,  tough  masses,  several  cm.  in 
diam.,  somewhat  similar  to  the  Ascococcus  Bitlrothii. 
They  cause  fermentation  in  solutions  of  dextrine, 
and  more  actively  in  potato  or  bean  paste.  Some 
cells  give  the  iodine  reaction  weakly,  as  in  Clostri- 
dium butyricum. 

Clostridium  of  symptomatic  anthrax  (Rausch- 
brand,  Charbon  symptomatigue.*)—R.o&s  rounded  at 
the  ends,  mostly  with  a  shining  spore  at  one  end. 
They  are  especially  distinguished  from  the  bacilli 
of  anthrax  by  being  motile.  Cultivated  on  blood- 

*  Arloing,  Cornevin  et  Thomas,  Bull,  de  VAcad.  de  Med.     1881. 


SYSTEMATIC    AND    DESCRIPTIVE.  321 

serum,  threads  develop,  consisting  of  both  rods  and 
cocci.  From  blood-serum  they  can  be  cultivated 
on  nutrient  gelatine,  and  vegetable  albumen. 

Cultivation  does  not  deprive  the  micro-organism 
of  its  virulence,  but  heating  the  spores  to  85°  C. 
renders  them  harmless. 

Inoculation  in  the  subcutaneous  tissue  of  guinea- 
pigs,  rabbits,  calves,  and  sheep  proves  fatal. 
White  rats,  dogs,  and  fowls  have  an  immunity. 
Injection  into  the  veins  in  small  quantity  produces 
a  febrile  disorder,  in  larger  quantities  death. 
Animals  in  the  former  case  suffer  an  abortive  ill- 
ness, which  protects  them  against  further  inocula- 
tion. The  micro-organism  is  the  cause  of  a  disease 
in  cattle ,  * '  blackleg , ' '  "  quarter- evil, "  or  *  *  Rausch- 
brand"  At  the  autopsy  the  micro-organisms  are 
found  in  the  subcutaneous  connective  tissue,  in 
the  lymph  glands,  kidneys,  spleen,  and  lungs. 
An  irregular  tumour  is  formed  in  the  skin,  which 
develops  rapidly,  and  gives  crepitus  on  palpation. 
The  tumour,  which  is  haemorrhagic  effusion,  occur- 
ring in  the  extremities,  impedes  the  animal's  move- 
ments. The  cattle  infected  die  in  thirty-six  to 
forty-eight  hours. 

GROUP  IIL     LEPTOTRICHE^E. 

Genus      I.  Crenothrix. — Threads     articulated ;      cells 

sulphurless  ;  habitat  water. 
Genus    II.  Beggiatoa. — Threads   unarticulated  ;    cells 

with  sulphur  granules ;    habitat  water. 

21 


322  BACTERIOLOGY. 

Genus  III.  Phragmidiothrix. — Threads  jointless  ;  suc- 
cessive subdivision  of  cells  is  continuous  ;  cells 
sulphurless  ;  habitat  water. 

Genus  IV.  Leptothrix. — Threads  articulated  or  unar- 
ticulated  ;  successive  subdivisions  of  cells  not 
continuous  ;  cells  sulphurless. 

Genus  I. — Crenothrix. 

SPECIES. 

UNASSOCIATED  WITH  DISEASE  :— 

Crenothrix  Kiihniana     .         .     Simple  saprophyte. 

Crenothrix  Kiihniana,  Rabenhorst. — Cocci, 
rods,  and  thread-forms.  The  cocci  are  globular, 
i — 6  p,  in  diam.  The  threads  are  colourless,  1*5 — 
5  /A  thick,  and  club-shaped  at  the  extremity,  reach- 
ing a  diam.  of  6 — 9  fi.  The  threads  form  colonies 
with  a  brick-red,  olive-green,  or  dark-brown  to 
brown-black  coloration  caused  by  impregnation 
with  oxide  of  iron.  The  threads  are  distinctly  ar- 
ticulated, and  ensheathed.  The  segments  are  set 
free  when  the  sheath  bursts,  and  develop  into  new 
threads.  In  other  cases  the  segments  remain  en- 
closed, and  subdivide  into  discs,  which,  by  vertical 
fission,  break  up  into  globular  forms  (cocci).  These 
again  develop  into  new  threads,  either  within  the 
sheath  eventually  penetrating  it,  or  after  they  are 
set  free  (Fig.  125). 

The  micro-organism  appears  in  little  whitish  or 
brownish  tufts  in  wells  and  drain-pipes,  and  it  not 
only  renders  drinking-water  foul,  but  may  stop  up 
the  narrower  pipes. 


SYSTEMATIC    AND    DESCRIPTIVE. 


323 


FIG.  125.— CRENOTHRIX  KUHNIANA. 

0,  b>  c,  d,  e.  Cocci  in  various  stages  of  fission,  X  600. 

/.  Zoogloea  of  cocci,  X  600. 

g.  Various  forms  of  zooglcea,  natural  size. 

h.  Colony  of  threads  composed  of  rods  grown  out  of  a  zoogloea  of  cocci. 

* — r.  Thread-forms  ;  some  straight,  others  spiral,  with  more  or  less  differ- 
entiation between  base  and  apex,  (r)  is  composed  of  short  rods  at  the  base 
and  above  these  of  cylindrical  segments,  and  at  the  apex  these  segments  have 
divided  into  cocci,  X  600.  •  [After  Zopf.] 


324  BACTERIOLOGY. 

Genus  II. — Beggiatoa. 

SPECIES. 

UNASSOCIATED  WITH  DISEASE  : — 

Beggiatoa  alba    ....  Simple  saprophyte. 
Beggiatoa  mirabilis  „  „ 

Beggiatoa  roseopersicina      .         .  Chromogenic  saprophyte. 

Beggiatoa  alba,  Vauch. — Cocci,  rods,  spirals, 
and  threads  (Fig.  1 26).  The  threads  are  longer  and 
thicker  than  leptothrix,  indistinctly  articulated, 
actively  oscillating,  and  colourless  ;  their  protoplasm 
contains  numerous  strongly  refractive  granules 
consisting  of  sulphur.  They  occur  as  greyish-  or 
chalk-white  gelatinous  threads,  3 — 3*5  p  thick,  in 
sulphur  springs  and  marshes. 

Beggiatoa  mirabilis,  Cohn. — Threads  dis- 
tinguished from  others  of  this  genus  by  their 
breadth,  which  may  reach  30  p.  They  are  motile, 
bent  and  curled  in  various  ways,  and  rounded  at 
the  ends.  Around  the  threads  isolated  cells  have 
been  observed,  "  macrococci,"  but  spiral  forms  are 
as  yet  unknown.  The  threads  are  filled  with 
sulphur  granules.  They  occur  on  sea  water,  form- 
ing a  white  gelatinous  scum  on  decomposing 
algae,  etc. 

METHODS    OF    EXAMINING    SPECIES    OF    BEGGIATOA. 

The  articulation  of  the  threads  is  best  demonstrated 
by  staining  with  an  alcoholic  solution  of  methyl-violet, 
fuchsine,  or  vesuvin  ;  or  by  treating  with  sodic  sulphate, 
or  warm  glycerine. 


SYSTEMATIC    AND    DESCRIPTIVE. 


325 


Beggiatoa  roseo-persicina  (Cohnia  roseo-persi- 
cina.  Bacterium  rubescens,  or  Peach  -  coloured  bacterium , 
Lankester). — Cocci,  rods,  spirals,  and  threads  (Fig. 


FIG.  126. — BEGGIATOA  ALBA. 

A.  Threads,  at  base  distinctly  linked,  partly  spiral.  B.  A  thread,  spiral  in 
its  whole  length.  C,  D.  Fragments  detached  from  threads ;  immotile. 
E.  Active  spirillum-forms,  with  a  flagellum  at  either  end.  F.  G.  Thin 
and  short  spiral  forms.  H.  A  spiral  showing  the  individual  links.  X 
540.  [After  Zopf.] 

127).      The  cocci,  globular  or  oval,  reach  2*5  /*  in 
diam.     They  form  at  first  solid  families,  bound  to- 


326 


BACTERIOLOGY. 


gether  by  gelatinous  substance.  Later  they  become 
larger,  globular  or  ovoid  in  shape,  and  hollow,  con- 
taining watery  fluid  in  their  interior.  The  families 
reach  a  diameter  of  660  /x,  in  which  the  cocci  form 
simply  a  peripheral  layer.  The  hollow  families  or 
vesicles  are  often  perforated,  presenting  a  delicate 
reticulated  appearance,  which  finally  may  become 
broken  up  into  irregular  structures.  The  red  colour- 


. 


y 


FIG.  127. — SEVERAL  PHASE-FORMS  OF  BEGGIATOA  ROSEO-PERSICINA. 
[After  Warming.] 

ing  matter  can  be  distinguished  from  other  red 
pigments,  and  it  is  designated  by  the  name  bacterio- 
purpurin.  It  is  quite  distinct  from  the  pigment 
produced  by  Bacterium  ptodigiosum,  being  peach- 
blossom  red,  and  insoluble  in  water,  alcohol,  etc. 
Examined  spectroscopically  it  shows  a  strong  ab- 


SYSTEMATIC    AND    DESCRIPTIVE.  327 

sorption  in  the  yellow,  and  a  weaker  band  in  the 
green  and  blue,  as  well  as  a  darkening  in  the  more 
refrangible  half  of  the  spectrum.  In  the  cocci, 
especially  of  the  older  vesicles,  dark  granules  are 
to  be  seen,  which  consist  of  sulphur.  The  micro- 
organisms occur  on  the  surface  of  marshes,  or  on 
water  in  which  algse  are  rotting.  They  form  a 
rose-red,  blood-red,  violet-red,  or  violet-brown 
scum  ;  and  sometimes  in  such  quantity  that  whole 
marshes  and  ponds  may  be  coloured  blood-red  by 
them. 

All  the  following,  hitherto  described  as  distinct  species^ 
are  probably  only  phase- forms  of  Beggiatoa  roseo-per- 
sicina. 

Spirillum  sanguineum,  Cohn  (Ophidomonas 
sanguined). — Threads  3  /JL  and  more  in  thickness  with 
2 — 2\  spirals,  each  9 — 12  /*  high,  with  their  ends  provided 
with  fiagella.  Their  colour  is  due  to  the  presence  of  red- 
dish granules  contained  in  the  cells.  They  were  observed 
in  brackish  water  with  putrefying  substances. 

Spirillum  rosaceum,  Klein — Resembles  Spirillum 
undula,  but  is  reddish  in  colour;  the  colouring  matter  is 
insoluble  in  water,  alcohol,  or  chloroform. 

Spirillum  violaceum,  Warming. — Threads,  cres- 
cent-shaped, or  possessing  1}  or  i  spiral,  with  their  ends 
broad,  rounded,  and  provided  with  flagella.  The  colour  is 
due  to  the  contents,  which  are  violet. 

Monas  vinosa. — Round  or  oval  cells  of  about  2*5  p 
in  diameter,  often  united  in  pairs.  Their  motion  is  slow 
and  tremulous,  and  the  cell  substance  pale-red,  with  dark 
grains  interspersed.  Flagella  have  not  been  observed.  They 
were  observed  in  water  with  decaying  vegetable  matter. 


328  BACTERIOLOGY. 

MonaS  Okenii. — Short  cylindrical  cells,  5  p  wide, 
8 — 15  p  long,  with  rounded  ends.  They  undergo  lively 
movements,  each  end  being  provided  with  a  flagellum 
twice  as  long  as  the  cell  itself.  They  have  pale- red  cell- 
substance  with  dark  grains.  They  occur  in  stagnant 
water. 

Rhabdomonas  rosea. — Spindle-form  cells,  3-8— 
S'O  fi  broad,  20 — 30  /*  long.  They  exhibit  slow,  trembling 
movements,  having  at  each  end  of  the  cell  a  flagellum. 
The  cell  substance  is  very  pale,  with  dark  grains  inter- 
spersed. In  brackish  water. 

MonaS  Warmingii. — Cylindrical  cells,  rounded  at 
the  ends;  15  //-  long,  5 — 8  Abroad.  They  are  possessed 
of  a  flagellum  at  each  end  of  the  cell,  and  exhibit  rapid, 
irregular  movements.  The  cell  substance  is  pale- red,  and 
studded  at  the  rounded  ends  with  dark-red  grains. 

Gen  u  s  III. — Ph  ragm  idiot h  rix. 

SPECIES. 

UNASSOCIATED  WITH  DISEASE  : — 

Phragmidiothrix  multiseptata     .         .     Simple  saprophyte. 

Phragmidiothrix  multiseptata. —  Cocci  and 
threads.  The  latter,  3 — 6  p.  in  breadth,  are 
separated  by  transverse  partitions  into  short 
cylindrical  discs,  whose  height  is  a  fourth  or 
sixth  of  their  breadth.  Repeated  transverse  and 
longitudinal  division  takes  place  in  the  discs, 
resulting  in  the  formation  of  cocci.  The  cocci 
have  not  been  observed  isolated  from  the 
threads  in  a  free  state,  but  they  develop  in  situ 
into  slender  threads.  In  addition  to  this  continuous 
subdivision,  Phragmidiothrix  differs  from  Beggiatoa 


SYSTEMATIC    AND    DESCRIPTIVE.  329 

in  the  absence  of  sulphur,  and  from  Crenothrix  by 
its  wanting  a  sheath.  They  occur  attached  to  crabs 
(Gammarus  locusta)  in  sea  water. 

Genus  IV. — Leptothrix. 
SPECIES. 

ASSOCIATED  WITH  DISEASE  : — 

Leptothrix  buccalis         .         .         .     Saprophytic. 
Leptothrix   gigantea       ...  ,, 

Leptothrix  buccalis,  Robin.  —  Long,  thin 
threads,  "j — i  p,  broad,  colourless,  often  united  in 
thick  bundles  or  felted  together.  Masses  of  cocci 
occur  with  the  threads,  and  the  threads  themselves 
are  composed  of  long  rods,  short  rods,  and  cocci. 
The  threads  may  break  up  into  spiral-,  vibrio-,  and 
spirochseta-forms.  The  last-named  occur  in  large 
numbers  in  the  mouth,  and  have  been  named  Spiro- 
chcete  buccalis.  The  Leptothrix  buccalis  is  found 
in  teeth  slime,  and  is  believed  to  be  intimately 
connected  with  dental  caries.  The  threads  pene- 
trate the  tissue  of  the  teeth,  after  the  enamel  has 
been  acted  upon  by  acids  generated  by  the  fer- 
mentation of  food.  The  short  rods,  long  rods, 
cocci,  leptothrix-forms,  and  screw-forms  are  found 
in  the  dental  canals. 

METHODS    OF    STAINING    THE    LEPTOTHRIX    BUCCALIS. 

The  threads  of  Leptothrix  buccalis  have  a  special  staining 
reaction  (Leber).  They  become  coloured  if  in  an  acid 
medium  with  iodine ;  if  the  medium  is  alkaline,  it  must  be 


33O  BACTERIOLOGY. 

acidified  with  very  dilute  hydrochloric  acid  or  acetic  acid, 
and  the  filaments  then  stained  with  iodine.  The  contents 
are  stained  violet,  and  contrast  with  the  sheath  and  septa, 
which  remain  uncoloured. 

Leptothrix  gigantea,  Miller.  —  Long  rods, 
short  rods,  and  cocci,  can  be  observed  in  the  same 
thread.  There  are  also  screw-threads,  which  may 
take  the  form  of  spirals,  vibrios,  or  spirochsetse. 
The  threads  increase  in  diameter  from  base  to 
apex,  and  corresponding  with  the  thickness  of 
the  threads,  the  rods  and  cocci  show  different 
dimensions.  They  have  been  observed  in  the 
diseased  teeth  of  dogs,  sheep,  cats,  and  other 
animals. 


GROUP  IV.     CLADOTRICHE^E. 

Genus  L — Cladothrix. 
SPECIES. 

UNASSOCIATED  WITH  DISEASE  : — 

Cladothrix  dichotoma  .         .         .     Saprophytic. 

Cladothrix  Foersteri      ...  ,, 

ASSOCIATED  WITH  DISEASE  : — 

Actinomyces          .         .         . x       .     Pathogenic. 

Cladothrix  dichotoma,  Cohn.  — Threads  re- 
sembling those  of  leptothrix;  slender,  colourless, 
not  articulated,  straight  or  slightly  undulated,  and 
in  places  twisted  in  irregular  spirals  with  pseudo- 
branchings.  The"  development  can  be  traced 
from  the  cocci  to  rods  and  threads.  The  latter 
are  at  the  beginning  simple  threads,  which  were 


SYSTEMATIC    AND    DESCRIPTIVE. 


331 


formerly  described   as  Leptothrix  parasitica,    or    if 
coloured  by  impregnation  with  iron,  as  Leptothrix 


FIG.  128. — CLADOTHRIX  DICHOTOMA. 

A.  Branching  schizomycete  :   (a)  Vibrio-form  ;    (o)    Spirillum-form   [slightly 

magnified]. 

B.  A  screw-form  with  (a)  Spirillum-form  ;  (b)  Vibrio-form. 

C.  Long  spirochseta-form. 

D.  Fragment  with  spirillum-form  at  one  end.  vibrio-form  at  the  other. 

E.  Screw-forms  ;    (a)  continuous ;    (b}  composed   of  rods ;  (c )  composed    of 

cocci. 

F.  Spirochseta-form  :   (a)  continuous ;  (b}  composed  of  long  rods  ;   (c)  short 

rods  ;  (d)  cocci.     [After  Zopf.] 

ochracea.      Later     they     form     false    branches    by 
single     rods    turning    aside,     which    by    repeated 


332  BACTERIOLOGY. 

division  lengthen  into  threads.  A  thread  appears 
to  be  first  composed  of  long  rods,  then  of  short 
rods,  and  lastly  of  cocci.  The  iodine  reac- 
tion must  be  applied  to  distinguish  these  forms, 
especially  when  the  sheath  of  the  threads  has 
a  yellow,  rust-red,  olive-green,  or  dark-brown 
coloration.  The  cocci  may  grow  into  rods  while 
still  in  the  sheath,  and  finally  become  leptothrix 
threads,  surrounded  by  a  delicate  gelatinous 
sheath,  from  which  the  false  branching  proceeds. 
Fragments  may  break  off,  which  are  actively 
motile,  and  appear  as  vibrios,  spirilla,  and  spiro- 
chseta-forms  (Fig.  128).  They  may  also  occur  in 
zooglcea. 

They  are  the  commonest  of  all  bacteria  in 
both  still  and  running  water,  in  which  organic 
substances  are  present.  They  are  observed  also 
in  the  waste  water  of  certain  manufactures,  such 
as  sugar.  Artificially  they  can  be  cultivated  on 
infusions  of  rotting  algse  and  animal  substances, 
forming  on  these  media  small  tufts,  about  i — 3  /*, 
and  floating  masses. 

Cladothrix  Fcersteri  (Streptothrix  Forsteri, 
Cohn). — Cocci,  rod-forms,  and  leptothrix-threads. 
The  threads  are  twisted  in  irregular  spirals, 
and  branch  sparingly  and  irregularly.  Screw- 
forms  are  produced  by  the  threads  breaking  up 
into  small  pieces.  They  occur  in  the  lachrymal 
canals  of  the  human  eye,  in  the  form  of  closely 
felted  masses. 


SYSTEMATIC    AND    DESCRIPTIVE.  333 

Here  we  may  add  some  little- known  species,  which 
possibly  belong  to  this  group. 

Sphaerotilus  natans. — Cells  4— 9  /t  long,  3  //,  thick, 

united  in  a  gelatinous  sheath  to  form  threads.  The  cells 
comprise  rods  and  cocci-forms  ;  the  cocci  are  set  free,  and 
develop  into  rods,  which  again  form  threads.  In  the  last  a 
false  branching  has  been  observed.  The  plasma  of  the 
cells  breaks  up  into  minute,  strongly  refractive  portions, 
which  develop  into  round  spores,  at  first  of  a  red,  and 
afterwards  a  brown  colour.  They  occur  in  stagnant  and 
flowing  water  contaminated  with  organic  matter,  and  form 
floating  flakes  of  a  white,  yellow,  rust-red,  or  a  yellow- 
brown  colour. 

MyconostOC  gregarium,  Cohn. — The  threads  are 
very  thin,  colourless,  unarticulated,  but  fall  apart  into  short 
cylindrical  links  when  dried.  They  form  gelatinous  masses, 
10 — 17  //,  in  diameter,  singly  or  heaped  into  slimy  drops  on 
water  in  which  algse  are  decomposing. 

Spiromonas  volubilis,  Perty. — Colourless,  trans- 
parent cells,  15  —  1 8  //,  long.  Rapidly  motile  and  revolving 
round  a  longitudinal  axis.  They  occur  in  marsh  water  and 
putrefying  infusions. 

Spiromonas  Cohnii. — Colourless  cells,  consisting 
of  i^  spirals,  with  both  ends  acutely  pointed  and  provided 
with  a  flagellum.  Breadth  of  the  cells  1*2 — 4  p.  They 
occur  in  water  containing  decomposing  matter. 

The  following  species  is  described  last,  so  that  it  may 
stand  between  the  cladothrix  group  of  bacteria  and  the 
hyphomycetous  fungi  given  in  the  appendix  which 
follows.  It  is  attached  provisionally  to  the  former  for 
reasons  stated  below,  but  there  is  need  for  further  inves- 
tigation before  its  position  is  established,  as  there  is  still 
some  doubt  as  to  the  true  life-history  of  the  fungus. 


334  BACTERIOLOGY. 

There  is  also  reason  for  believing  that  there  are 
different  forms  of  actinomy costs  in  animals •,  and  that 
the  rare  disease  in  man  differs  etiologically  from  the 
not  so  uncommon  disease  in  cattle.  It  is  only  then  pro- 
visionally that  the  different  forms  are  here,  described 
under  one  heading. 

Actinomyces.  —  Actinomycosis  is  a  disease 
occurring  in  animals  *  and  occasionally  in  man.t 
It  is  caused  by  a  parasite  known  as  Actinomyces, 
or  the  "  ray-fungus."  The  parasite  appears  in  the 
form  of  a  rosette  of  pyriform  or  club-shaped  ele- 
ments (Plate  XXIX.,  Fig.  i).  The  little  masses  are 
colourless,  pure  white,  or  of  a  yellowish  or  yellowish- 
green  tinge,  and  visible  to  the  naked  eye. 

The  fungus  is  believed  to  effect  an  entrance  to 
the  animal  by  the  mouth,  being  taken  in  with  the 
food,  possibly  through  the  medium  of  a  wound  of 
the  gum  or  a  carious  tooth.  In  whatever  manner 
it  has  gained  access  to  the  living  organism,  it  sets 
up  inflammation  in  its  neighbourhood,  resulting 
in  the  formation  of  a  neoplasm,  composed  chiefly 
of  round  cells,  resembling  a  tuberculous  nodule. 
The  nodules  may  break  down  and  suppurate,  or 
may  go  on  increasing  in  size.  Fibrous  tissue  develops 
between  the  nodules,  and  large  tumours  eventually 
result  containing  purulent  cavities  and  excavations. 

*  Bellinger,   Centralbl.  f.  Med.  Wiss.     1877. 

f  Israel,  Virchow 's  Archiv,  vols.  74 — 78.  Pontick,  Die  Actino- 
mycose  des  Menschen.  1882  ;  and  Beitr.  z.  Kenntn.  der  Actino- 
mycose  des  Menschen.  1882  ;  Lancet,  May  2nd,  1885  >  Conti,  Gaz. 
Med.  ItaL  Lombordia*  1885  ;  Acland,  Trans.  Path.  Soc.  1886. 


SYSTEMATIC    AND    DESCRIPTIVE.  335 

In  the  slimy  detritus  the  little  pale-yellow  grains 
of  fungus  can  be  detected.  In  cattle  the  lower 
jaw  is  usually  affected,  and  then  the  upper  jaw 
and  neighbouring  parts.  The  organism  may  also 
occur  in  nodular  tumours  in  the  lung,  subcutaneous 
and  intermuscular  tissues.  It  is  the  cause  of 
"  wooden  tongue,"  and  also  of  diseases  which  have 
been  variously  described  before  their  true  nature 
was  understood  as  bone-canker,  bone-tubercle, 
osteo-sarcoma,  etc. 

In  man  the  pulmonary  formations  tend  to 
break  down  early,  forming  fistulae  and  sinuses, 
with  the  clinical  character  of  empyema.  In  one 
case  there  were  the  symptoms  of  chronic  bronchitis 
with  foetid  expectoration.  In  other  cases  the  dis- 
ease, originating  in  the  lung,  spread  to  the  prse- 
vertebral  tissues.  If  the  actinomyces  invade  bones, 
as  has  been  especially  observed  in  the  bodies  of  the 
vertebrae,  caries  results.  In  another  group  of  cases 
the  organism  has  been  said  to  produce  disease  of 
the  intestinal  canal.  The  fungus  has  also  been 
detected  in  the  crypts  of  the  tonsils  of  healthy  pigs, 
and  a  similar,  if  not  identical,  one  in  the  sper- 
matic duct  of  the  horse.* 

The  disease  has  been  transmitted  from  cattle  to 
cattle  by  inoculation,")"  and  a  rabbit  has  been  in- 
fected by  means  of  a  piece  of  actinomycitic  tumour 

*  Johne,    Bericht    uber    das    Veterinarwesen   im  Konigreich 
Sachsen  fur  das  Jahr.    1884. 
t  Johne,  Deutsche  Zeitschr.  f.  Thier Median.     1881. 


336  BACTERIOLOGY. 

from  a  human   subject,    introduced  into    the  peri- 
toneal cavity. 

Until  quite  recently  actinomyces  has  been  classed 
as  a  hyphomycete,    and  the  flask-shaped  structures 
regarded    as    gonidia.       By    certain  *    cultivation- 
experiments  we  are  led  to  regard  the  latter  as  a 
result  of    a   degenerative  stage  in  the  life-history 
of   the   fungus  accompanied    by   the   development 
of    involution  -  forms.      Inoculations     of    nutrient 
gelatine,    in    the    form    of   plate-cultivations,  and 
inoculations   on   blood    serum    and   nutrient   agar- 
agar  were  made,    it  is  stated,  with  success.     The 
cultures  developed  on  the  latter  in  from  five  to  six 
days,  growing  best  at  a  temperature  of  33° — 37°  C. 
Nutrient  gelatine  was  not  liquefied.     The  appear- 
ances   of  the  cultivation  were  described  as  quite 
characteristic  ;    it  has  at   first  a  whitish,  granular 
appearance,    followed    after   a   few   days   by   little 
yellowish-red  spots  which   coalesce  in    the  centre, 
and  finally  a   whitish    downy  layer  results  with  a 
golden- red    centre ;    in    time    the    periphery    also 
becomes  dotted  with  little   yellow-centred  masses. 
The  fungus  thus  cultivated  has  been  described  as 
corresponding  on  examination  with  the  form  found 
in   man  and  animals,  and  at  one  stage  to  consist 
of    thread-forms,    short   rods,    and    cocci.       As    a 
result  of  these  observations  actinomyces  has  been 
relegated  to  the  bacteria,  forming  one  of  the  cla- 

*  Bostrom,   "  Ueber  Actinomycose,"     Verhandlungen   des  Con- 
g  r  esses  fur  Inn .  Med.     1885. 


SYSTEMATIC   AND    DESCRIPTIVE,  337 

dothrix   group,  and  possibly  closely  allied  to  the 
Streptothrix  Farsieri  of  Cobn. 

METHODS    OF    EXAMINING    AND    STAINING 
ACTINOMYCES 

In  the  fresh  state  a  little  of  the  tissue  of  a  tumour,  or  the 
purulent  detritus,  may  be  transferred  to  a  clean  glass  slide, 
and  teased  out  with  needles.  The  little  specks  are  easily 
recognised,  and  can  be  isolated  with  the  needles  and 
transferred  to  a  drop  of  glycerine  upon  a  fresh  slide.  A 
cover-glass  must  then  be  gently  pressed  down  upon  the 
preparation,  which  is  then  examined.  To  stain  the  fresh 
tufts,  the  little  fungus  masses  are  teased  out,  and  transferred 
to  a  watch-glass  containing  alcohol,  to  which  a  few  drops  of 
concentrated  alcoholic  solution  of  eosin  are  added.  They 
can  be  mounted  and  preserved  in  glycerine. 

Sections  can  be  stained  by  either  of  the  following 
methods. 

Weigerfs  Method: — 

Immerse  the  sections  for  one  hour  in  orseille,  rinse  with 
alcohol,  and  after-stain  with  gentian  violet  (Plate  XXVIII.). 

Plants  Method:— 

Sections  are  left  for  ten  minutes  in  Gibbes'  solution  (No. 
22),  warmed  to  45°  C.  They  are  then  rinsed  in  water,  and 
after-stained  in  concentrated  alcoholic  solution  of  picric 
acid  for  five  to  ten  minutes  ;  immersed  in  water  five  minutes, 
50  per  cent,  alcohol  fifteen  minutes,  passed  through  absolute 
alcohol  and  clove-oil,  and  preserved  in  Canada  balsam 
(Plate  XXVIL). 


22 


DESCRIPTION    OF     PLATE    XXIX. 


FIG. 
i . — Actinomyces,  teased  out  in  the  fresh  state  and  stained 

with  eosin. 

2. —  Torula  cerevisice  (after  Rees). 
3. — Saccharomyces  mycoderma,  or  oidium  albicans,   from 

an  artificial  cultivation  (after  Grawitz). 
4. — Saprolegnia  (after  Sachs). 
5.  —  Oidium  lactis  (after  Flugge). 

6. — Fungi  of  favus,  or  oidium  lactis  (after  Neumann). 
7. — Penicillium  glaucum  (after  Flugge). 
8. — Aspergillus   niger,   from   a  preparation    mounted    in 

glycerine. 
9. — Aspergillus  niger,  from   the  same  preparation  (Zeiss 


1    o.  i). 


Tf 

i  o. — Aspergillus  glaucus  (after  De  Bary). 
i  i. — Botrytis  Bassiana  (after  De  Bary). 


APPENDIX    A. 

YEASTS     AND     MOULDS. 

Yeast  -fungi  and  mould-fungi,  like  bacteria  or  fission- 
fungi^  are  achlor  aphyllous  Thallophytes.  They  belong  to 
two  separate  orders,  the  Saccharomycetes  and  Hyphomy- 
cetes,  which  are  intimately  related  to  each  other,  but  quite 
distinct  from  bacteria.  Their  germs  occur  widely  dis- 
tributed in  air,  soil,  and  water,  and  are  constantly  en- 
countered in  bacteriological  investigations.  In  addition 
many  species  are  of  hygienic  and  pathological  interest  or 
importance  in  being  either  accidentally  associated  with, 
or  actually  the  cause  of  various  morbid  processes.  For 
a  complete  account  of  all  the  described  species  and  full 
details  of  the  various  forms  of  development,*  reference 
must  be  made  to  botanical  treatises.  A  description  of 
certain  species  is  appended  here,  and  may  afford  some 
useful  information  to  the  worker  in  a  bacteriological 
laboratory. 

YEAST-FUNGI    OR    SACCHAROMYCETES. 


Saccharomyces  cerevisise  (Twula 
Cells  round  or  oval,  8  —  9  /*,  long,  singly  or  united  in 
small  chains.  Spores  occur  three  or  four  together  in  a 
mother-cell,  4  —  5  p,  in  diameter  (Plate  XXIX.,  Fig.  2).  ' 

Sacch.  ellipsoideus.  —  Elliptical  cells,  mostly  6  p. 
long,  singly  or  united  in  little  branching  chains.     Two  to 

*    Sachs,  Text-book  of  Botany.     1882. 


APPENDIX.  341 

four  spores  found  in  a  mother-cell,  3 — 3*5  p,  in  diam.  It  is 
widely  distributed,  and  is  the  principal  agent  in  accidental 
fermentation. 

SaCCh.  COnglomeratUS. — Cells  round,  united  in 
clusters,  consisting  of  numerous  cells  produced  by  budding 
from  one  or  a  few  mother-cells.  There  are  2  to  4  spores 
in  each  mother-cell.  They  occur  on  rotting  grapes  and 
in  wine  at  the  commencement  of  fermentation. 

Sacch.  exigUUS. — Conical  or  top-shaped  cells,  5  p, 
long,  and  reaching  2*5  fi  in  thickness,  in  slightly  branching 
colonies.  Spore-forming  cells  are  isolated,  each  contain- 
ing 2  or  3  spores  in  a  row.  Present  in  the  after-fermenta- 
tion of  beer. 

Sacch.  pastorianus. — Cells  oval  or  club-shaped. 
Colonies  consist  of  primary  club-shaped  links,  18 — 
22  p,  long,  which  build  lateral,  secondary  round  or  oval 
daughter-cells,  5 — 6  p,  long.  Spores  2  to  4.  In  the  after- 
fermentation  of  wine,  fruit-wines,  or  fermenting  beer. 

Sacch.  apiculatus. — Cells  lemon-shaped,  both 
ends  bluntly  pointed,  6 — 8  p,  long,  2  —  3  //,  wide. 
Budding  occurs  only  at  the  pointed  ends.  Rarely  united 
in  colonies.  Spores  unknown.  They  occur  with  other 
yeasts  in  various  accidental  fermentations. 

Sacch.  Sphsericus. — Cells  varying  in  form  ;  the 
basal  ones  of  a  colony  oblong  or  cylindrical,  10 — 15  JJL 
long,  5  //,  thick;  the  others  round,  5 — 6  p,  in  diam. 
United  in  ramified  families.  Spores  unknown. 

Sacch.  mycoderma  (Mycodernta  cerevisicz  et  vini). 
— Cells  oval,  elliptical,  or  cylindrical,  6 — 7  JJL  long,  2  —  3  p, 
thick,  united  in  richly-branching  chains.  Spore-forming 
cells  reaching  20  //,  long.  Spores  i  to  4  in  each  mother- 
cell.  Forms  the  so-called  "  mould  "  on  fermented  liquids, 
and  develops  on  the  surface  without  exciting  fermenta- 
tion. When  forced  to  grow  submerged,  a  little  alcohol  is 
produced,  but  the  fungus  soon  dies.  They  occur  on  wine, 
beer,  fruit-juices,  and  sauerkraut. 

Sacch.  albicans  (Oidium    allicans}. — Cells    partly 


342  APPENDIX. 

round,  partly  oval  or  cylindrical,  35 — 5  /x,  thick,  the 
cylindrical  cells  10 — 20  times  as  long  as  they  are  thick. 
The  bud-colonies  mostly  consist  of  rows  of  cylindrical 
cells,  from  the  ends  of  which  oval  or  round  cells  shoot 
out.  Spores  form  singly  in  roundish  cells.  They  occur  on 
the  mucous  membrane  of  the  mouth,  especially  of  infants, 
in  greyish-white  patches  which  consist  of  epithelium, 
bacteria,  yeasts,  and  the  mycelia  of  various  moulds.  They 
can  be  easily  cultivated  in  a  nutrient  solution  containing 
sugar  and  ammonic  tartrate.*  The  cells  germinate  accord- 
ing to  the  richness  of  the  fluid  in  sugar  ;  they  either  grow 
into  long  threads,  or,  in  a  very  strongly  saccharine  solution, 
many  daughter-cells  are  formed,  budding  out  in  various 
directions  (Plate  XXIX.,  Fig.  3). 

Sacch.  glutinis. — Cells  round,  oval,  or  short 
cylinders,  5 — 1 1  JJL  long,  4  p,  wide,  isolated,  or  united 
in  twos.  Cell-membrane  and  contents  are  colourless  in 
the  fresh  state,  but  when  dried  and  remoistened  possess 
a  pale-reddish  nucleus  in  the  middle.  Spore  formation 
unknown.  Forms  rose-coloured,  slimy  spots  on  starch, 
paste,  or  on  sterilised  potatoes.  The  colouring  matter 
is  not  changed  by  acids  or  alkalies. 

Sacch.  rosaceus  (Pink  Torula). — Cells  9 — 10  /x 
in  diam.  Forms  a  coral-pink  growth  in  nutrient  gelatine, 
nutrient  agar-agar  (Plate  XIII.,  Fig.  3),  or  on  sterilised 
potatoes  (Plate  X.,  Fig.  2).  They  are  present  in  the 
air. 

Sacch.  niger  (Black  Torula). — Cells  also  present  in 
the  air.  Cultivated  in  nutrient  gelatine  they  form  a 
black  crust  (Plate  III.,  Fig.  3). 

MOULD-FUNGI      OR      HYPHOMYCETES. 

The  mould-fungi  have  been  divided  into  five  orders  :f 
Hypodermii,  Phy  corny  cetes,  Ascomycetes,  Basidiomycetes,  and 

*Grawitz,  Virch.  ArcJiiv,  vol.  70. 

|  Fliigge,  Fermente  u.  Mikrofiarasiten.     1883. 


APPENDIX.  343 

Myxomycetes.     The  following  species,  with  the  orders  to 
which  they  belong,  are  of  especial  interest. 


HYPODERMII. 

UstilagO  carbo  (mildew,  smut). — Spores,  brown, 
circular ;  episporium  smooth  ;  sporidia,  ovoid  cells.  The 
spores  or  conidia  occur  as  a  black  powder  in  the  ears  and 
panicles  of  wheat,  barley,  and  oats. 

Tilletia  caries. — Spores  round,  pale,  brown  ;  epi- 
sporium with  reticulated  thickenings.  In  germinating 
sporidia  grow  out  radially  from  the  end  of  the  promy- 
celium  ;  these,  at  their  lower  part,  conjugate  by  a  cross 
branch,  and  separate  from  the  promycelium,  and  at  some 
point  of  the  pair,  a  hypha  grows  out  on  which  abundant 
secondary  sporidia  develop.  The  latter  are  long,  oval 
cells,  which  can  in  turn  germinate.  The  fungus  occurs 
in  the  form  of  a  stinking  powder  in  grains  of  wheat, 
which  renders  the  meal  impure,  and  gives  it  a  disagreeable 
smell. 

Urocystis  OCCulta. — The  spores  consist  of  several 
cells  united  together  ;  partly  large,  dark-brown  cells  in  the 
interior,  and  outside  several  flat  semi-circular,  colourless 
cells.  Spores  "024  mm.  Promycelium  germinates  as  in 
Tilletia^  but  the  cylindrical  cells  produce  a  hypha,  without, 
as  a  rule,  previous  conjugation.  Occurs  as  a  black  powder 
in  rye  straw,  in  long  disintegrated  stripes,  which  are  at 
first  greyish.  The  affected  plant  produces  abortive  ears. 

Empusa  muscae. — Spores,  -on  mm.  in  diam. 
A  spore  or  conidium  alighting  upon  the  white  area  of 
the  under  surface  of  the  body  of  the  house-fly,  germinates 
into  a  hypha.  The  latter,  penetrating  the  skin,  forms  toru- 
Ibid  cells,  which  multiply  by  germination,  and  are  dissemi- 
nated in  the  blood  throughout  the  body  of  the  fly.  These 
cells  again  grow  into  hyphae,  which  penetrate  the  skin, 
each  forming  a  conidium,  which  is  cast  off  with  consider- 
able force.  The  parasite  is  fatal  to  flies,  especially  in  the 


344  APPENDIX. 

autumn.  They  are  often  observed  attached  to  the  walls 
or  window-panes,  surrounded  by  a  powdery  substance, 
consisting  of  the  extruded  conidia. 

Empusa  radicans. — The  spores  form  long  hyphae, 
which  pierce  the  transparent  skin  of  the  caterpillar  of  the 
cabbage-white  butterfly.  The  terminal  cells  ramify,  and 
fill  the  body  of  the  caterpillar  with  a  network  of  mycelial 
filaments.  The  caterpillars  attacked  become  restless,  then 
motionless,  and  death  ensues. 

Tarichium  megaspermum. — The  spores  are  -05 
mm.  in  diam.,  black  in  colour,  and  provided  with  a  thickened 
episporium.  They  occur  at  the  sides  and  ends  of  myce- 
lial threads,  attacking  caterpillars  (Agrotis  segetmtt). 

PHYCOMYCETES. 

Saprolegnia. — Colourless  threads,  forming  dense  radi- 
ating tufts,  occur  on  living  and  dead  animal  and  vegetable 
matter  in  fresh  water.  The  filaments  penetrate  into  the 
substratum,  and  branch  more  or  less  in  the  surrounding 
water.  The  cylindrical  ends  of  threads  are  shut  off  by 
a  septum  forming  zoosporangia,  or  mother-cells,  in  the 
interior  of  which  a  number  of  spherical  spores,  zoospores, 
develop.  These  are  set  free  through  an  apical  opening  in 
the  thread,  and,  after  a  time  coming  to  rest,  give  rise  to 
new  plants  (Plate  XXIX.,  Fig.  4).  In  the  sexual  mode 
of  reproduction,  a  spherical  bud,  the  oogonium,  deve- 
lops at  the  end  of  a  mycelial  thread  ;  from  the  thread, 
small  processes  or  antheridia  sprout  out  laterally  towards 
the  oogonium,  and  blend  with  its  protoplasm  (Plate  XXIX., 
Fig.  4).  The  latter  breaks  up  into  a  number  of  oospores, 
which  clothe  themselves  with  a  membrane,  while  still  within 
the  mother-cell,  and  eventually  being  set  free,  grow  into 
fresh  mycelial  filaments.  The  parasite  attacks  fish  and 
tritons,  and  produces  a  diseased  condition  of  the  skin,  which 
may  be  ultimately  fatal.  In  salmon  it  produces  the  com- 
mon disease  of  salmon. 

Peronospora  infestans. — Mycelium,  -005  mm.  in 


APPENDIX.  345 

thickness.  Twigs  with  as  many  as  five  branches,  each 
bearing  an  egg-shaped  conidium.  The  contents  of  the 
conidia  falling  off  and  reaching  a  drop  of  moisture,  break 
up  into  a  number  of  swarming  zoogonidia,  which  in 
turn  develop  upon  plants.  Fixing  themselves  to  the 
cuticle  of  the  host,  they  throw  a  germinating  filament  into 
an  epidermal  cell  ;  piercing  first  its  outer  wall,  and  then 
its  inner  wall,  the  filament  reaches  an  intercellular  space, 
where  the  mycelium  develops.  This  continues  to  grow 
and  spread  throughout  the  plant.  In  tubers  it  can  hiber- 
nate and  develop  in  the  young  shoots  in  the  following 
spring.  The  parasites  appear  in  the  form  of  brown 
patches  on  the  green  parts  of  the  plants,  especially  the 
leaves.  The  attacked  parts  wither  and  turn  yellow  or 
brown  in  colour.  If  the  under  surface  of  a  diseased  leaf 
be  examined,  a  corresponding  dark  spot  may  be  observed, 
accompanied  with  a  faint  greyish-white  bloom  which 
covers  it.  The  latter  consists  of  the  conidia-bearing 
branches  of  the  fungus. 

Pilobolus. — Hyphae,  I — 2  mm.  high.  Fruit-hyphae, 
possessing  spherical  receptacles  containing  conidia.  When 
ripe  the  receptacles  with  their  conidia  are  detached  at 
their  bases  and  spring  by  their  elasticity  to  some  distance. 
The  mould  occurs  as  glassy  tufts  on  the  excrement  of 
cows,  horses,  etc.  A  cultivation  can  generally  be  obtained 
by  keeping  fresh  horse-dung  under  a  bell-glass. 

Mucor  mucedo. — Hyphae,  colourless,  simple  or 
branched,  I  — 15  cm.  long,  sporangia  are  yellowish-brown 
or  black.  Spores  ovoid, '008  mm.  long,  and  "0037  wide. 
Occurring  as  the  familiar  white  mould  on  fruits,  bread, 
potatoes,  excreta,  and  penetrating  into  the  interior  of 
nuts  and  apples.  A  network  of  fibrils  develops  in  the 
substance  of  nutrient  gelatine,  with  formation  of  sporangia 
on  the  free  surface.  The  germination  of  the  spores  and 
development  into  hyphae  can  be  observed  in  a  few  hours, 
if  the  fungus  be  cultivated  in  a  decoction  of  horse  dung. 

Mucor  racemoSUS. — Hyphae,  at  most  I  '5  cm.  long 


346  APPENDIX. 

sporangia,  yellowish  to  pale-brown  ;  spores  round.  By 
continued  cultivation  in  liquids  saturated  with  carbonic 
acid,  the  hypha  becomes  shorter,  and  exhibits  a  yeast-like 
sprouting.  These  yeast-like  or  toruloid  cells  can,  when 
the  carbonic  acid  is  withdrawn,  germinate  into  normal 
mycelium. 

Mucor  Stolonifer,  Lichtheim. — Mycelium  grows 
in  the  air,  and  then  bends  down  and  re-enters  the  nutrient 
substratum  ;  sporangia  black,  and  spores  globular.  The 
mycelium  can  penetrate  through  the  shell  of  eggs,  and 
form  conidiophores  within  them. 

Mucor  aspergillus,  Lichtheim.  Fruit  hyphae, 
thinned  at  the  base,  and  with  many  fork-like  divisions, 
dark-brown  spores. 

Mucor  phycomyces,  Lichtheim. — Mycelium  thick- 
walled,  olive-green  fruit-hyphae,  black  sporangia,  and 
oblong  spores. 

Mucor  macrocarpUS, Lichtheim. — Spindle- formed, 
pointed  spores. 

Mucor  fusiger,  Lichtheim. — Ovoid  spores. 

Mucor  mellittophorus,  Lichtheim. — Spores  ellip- 
tical. Found  in  the  stomach  of  bees. 

Mucor  corymbifer,  Lichtheim. — Forms  branched 
fruit-hyphae  ;  sporangium  has  a  smooth  membrane. 
Found  in  the  external  auditory  meatus  ;  occurring  also 
upon  bread.  Pathogenic  in  rabbits. 

Mucor  rhizopodiformis,  Lichtheim. — Occurs  on 
bread.  The  spores  of  Mucor  rhizopodiformis  and  Mucor 
corymbifer,  when  introduced  into  the  vascular  system  of 
rabbits,  can  germinate  in  the  tissues,  especially  in  the 
kidneys,  where  they  set  up  haemorrhagic  inflamma- 
tion. Dogs  are  immune,  and  only  artificial  mycosis 
is  known.* 

*  Lichtheim,  Zeistchr.  f.  Klin.  Med.,  vii. ;  Hiickel,  Beitr.  z, 
Anat.  u.  Phys.y  herausgeg.  v.  Ziegler  u.  Nauwerck.  1885. 


APPENDIX.  347 

ASCOMYCETES. 

OidiumTuckeri. — Fruit  hyphae,  bearingsingle  ovoid 
conidia.  Observed  in  the  form  of  brown  patches,  covered 
with  a  white  mildew-like  layer  on  the  leaves,  branches,  and 
young  fruit  of  the  vine,  producing  a  "  grape-disease." 

Oidium  lactis. — Fruit  hyphae,  simple,  erect,  and 
colourless,  bearing  at  their  ends  a  series  or  chain  of 
conidia  (Plate  XXIX.,  Fig.  5).  In  some  cases  the  fruit 
hypha  branches  beneath  the  chain  of  spores.  Spores  are 
short  cylinders,  '0077 — "0108  mm.  long.  The  fungus 
is  sometimes  found  as  a  whitish  mould  on  milk,  bread, 
paste,  potato,  and  excrement,  and  is  believed  to  be  iden- 
tical* with  the  fungus  of  certain  human  skin  diseases, 
Favus  (Achorion  Schoenleinii}^  Herpes  tonsurans  (Tri- 
cophyton  tonsurans)  and  Pityriasis  versicolor  ( Microsporon 
furfur)  (Plate  XXIX.,  Fig.  6).  Cultivated  artificially  in 
nutrient  gelatine,  the  conidia  germinate  into  filaments  of 
varying  length,  which  by  subdivision  form  septate 
mycelial  hyphae  ;  these  and  their  branches  give  rise  in 
turn  to  spores  or  conidia.  The  differences  observed  in 
various  diseases  are  attributed  to  differences  in  the  nutrient 
substratum.  Others")"  maintain  that,  in  artificial  cultiva- 
tions of  the  spores  of  Tricophyton  tonsurans^  the  fructifica- 
tion is  identical  with  Penicillium. 

Oidium  albicans. — Vide  Saccharomyces  albicans. 

Aspergillus  glaucus  (Eurotium  aspergillus  glau- 
cus). — Mycelium,  at  first  whitish,  becoming  grey-green 
or  yellow-green.  Spores  grey-green,  thick-walled,  "009 
— "015  mm.  in  diam.  Sometimes  found  on  various 
substances,  chiefly  cooked  fruit  (Plate  XXIX.,  Fig.  10). 

Aspergillus  repens  (Eurotium  repens\  De  Bary. 
— Fruit  heads  fewer  than  in  the  above,  which  are  at  first 
pale  and  then  blue-green  to  dark-green  in  colour  ;  conidia 
mostly  oval,  smooth,  '005 — '008  mm.  long,  colourless  or 
pale  to  grey-green. 

*  Grawitz,  Vir chow's  Archiv,  vol.  70. 

+  Morris  and  Henderson,  Journ.  Royal  Microsc.  Society.     1883. 


348  APPENDIX. 

AspergillllS  flavus. — Gold-yellow,  greenish  and 
brown  tufts  ;  fruit  heads  round  ;  yellow,  olive-green,  or 
brown.  Conidia  round,  seldom  oval  ;  sulphur-yellow  to 
brown  in  colour,  '005 — "007  mm.  in  diam.  Saprophytic 
in  man,  pathogenic  in  rabbits. 

Aspergillus  fumigatus. — Greenish,  bluish,  or 
grey  tufts.  Fruit  heads  long  and  conical.  Conidia  round, 
seldom  oval,  smooth,  mostly  pale  and  colourless.  Diam. 
•0025  to  '003  mm.  Observed  saprophytically  in  human 
lungs,  external  auditory  meatus,  and  middle  ear.  The 
spores  introduced  into  the  vascular  system  of  rabbits,  or 
into  the  peritoneal  cavity,  establish  metastatic  foci  in  the 
kidneys,  liver,  intestines,  lungs,  muscles,  and  sometimes  in 
the  spleen,  bones,  lymphatic  glands,  nervous  system, 
and  skin. 

Aspergillus  niger  (Eurotium  aspergillus  niger,  De 
Bary). —  Dark  chocolate-brown  tufts.  Conidia  round, 
black-brown,  or  grey-brown,  when  ripe  ;  '0035  to  '005  mm. 
This  mould  can  be  cultivated  readily  on  bread  moistened 
with  vinegar,  on  slices  of  lemon,  and  on  acid  fruits  and 
liquids.  It  flourishes  best  of  all,  according  to  Raulin,* 
in  a  liquid  of  the  following  composition  :  — 

Grammes. 

Water 1500 

Sugar-candy.          .          .          .          .  70* 

Tartaric  acid          V        •          .          .  4* 

Nitrate  of  ammonia         ...  4 

Phosphate     ...          .          .  "6 

Carbonate  of  potassium  .          .          .  *6 

„            „   magnesium           .          .  -4 

Sulphate  of  ammonia      .          .          .  "25 

„           „  zinc               .          .          .  -07 

„  iron               ...  -07 

Silicate  of  potassium       .          .          .  "07 

It  was  also  found  that  the  fungus  grew  best  when  the 
liquid  was  spread  out  in  a  layer  2  or  3  cm.  in  depth  in  a 
*  Duclaux,  Health  Exhibition  Handbook,  London,  1884. 


APPENDIX.  349 

shallow  dish,  and  a  temperature  of  35°  C.  proved  to  be 
the  most  favourable.  The  abstraction  of  zinc  from  the 
nutritive  liquid  reduced  the  weight  of  a  crop  from  25  (the 
average)  to  2  grammes,  and  the  presence  of  Tfrtfo^Tny 
part  of  nitrate  of  silver,  or  wJcnr  part  of  corrosive 
sublimate,  stopped  the  growth  altogether.  It  is  sapro- 
phytic  in  the  living  body. 

METHOD    OF    EXAMINING   ASPERGILLUS    NIGER. 

Species  of  aspergillus  stain  intensely  with  carmine, 
fuchsine,  or  methyl-violet,  but  to  examine  A  spergillus  niger 
with  a  high-power,  a  little  special  technique  is  employed, 
as  follows  : — A  drop  of  glycerine  is  placed  on  a  clean 
slide,  and  a  drop  of  alcohol  on  a  cover-glass.  With  a 
fine  pair  of  forceps  a  few  of  the  fruit  hyphc'E  with  their 
black  heads  are  immersed  in  the  alcohol.  The  cover- 
glass  is  then  turned  over  on  to  the  drop  of  glycerine,  and 
the  slide  held  in  the  flame  of  a  Bunsen  burner  till  the 
spores  or  conidia  are  dispersed.  To  make  a  permanent 
preparation,  remove  the  cover-glass,  and  transfer  the  fruit 
hyphae  so  treated  to  a  mixture  of  glycerine  and  water 
(i  to  5)  ;  a  drop  may  be  conveniently  placed  ready  on  a 
slide  provided  with  a  ring  of  Canada  balsam.  The  speci- 
men is  then  permanently  mounted  by  employing  a  circular 
cover-glass,  and  surrounding  it  with  a  ring  of  cement  in 
the  usual  way  (Plate  XXIX.,  Figs.  8  and  9). 

Aspergillus  ochraceus. — At  first  flesh-coloured, 
and  then  ochre-yellow  heads. 

Aspergillus  albus. — Pure  white  fruit  heads. 

Aspergillus  clavatUS. — Club-shaped  fruit  heads 
on  long  stems. 

Penicillium  glaucum. — Occurs  as  a  white,  and 
later  a  blue-green  mould,  on  which  dewlike  drops  of 
liquid  may  appear  (Plate  IX.,  Fig.  2).  Its  spores  are 
present  in  large  numbers  in  the  air,  and  are  liable  to 
contaminate  cultivations.  Diam.  of  the  spores  '0035  mm-  J 
threads  vary  in  diameter  between  "004  and  '00071  mm., 


35O  APPENDIX. 

according  to  the  nourishing  material ;  the  fruit  hypha 
bears  terminally  a  number  of  branched  cylindrical  cells, 
from  which  chains  of  greenish  conidia  are  developed 
(Plate  XXIX.,  Fig.  7).  It  is  the  commonest  of  all 
moulds. 

Botrytis  Bassiana. — Hyphae  and  spores  colour- 
less. Hyphae  usually  simple,  but  sometimes  united  in 
arborescent  stems  (Plate  XXIX.,  Fig.  11),  It  is  the 
cause  of  muscardine,  a  fatal  disease  of  silkworms,  and 
occurs  also  in  various  other  caterpillars  and  insects. 

UNCLASSED. 

Chionyphe  Carter!. — Mycelium,  penetrating  the 
skin  and  subcutaneous  tissue,  sets  up  suppuration  and 
ulceration.  Described  as  the  cause  of  a  disease  known  in 
India  as  "  madura-foot." 


APPENDIX  B. 

FLAGELLATED  PROTOZOA  IN  THE  BLOOD.* 

WHEN  examining  blood  the  bacteriologist  must  be  pre- 
pared to  meet  with  minute  organisms  which  at  the  first 
glance  under  moderate  amplification  may  be  mistaken  for 
vibrionic  or  spiral  forms  of  bacteria.  The  organisms 
referred  to  belong  not  to  the  vegetable,  but  to  the  animal 
kingdom.  They  may  occur  associated  with  disease,  but 
they  appear  to  be  more  commonly  found  in  the  blood  of 
apparently  perfectly  healthy  animals. 

Flagellated  organisms  in  the  blood  of  rats 
and  hamsters. — Lewisj*  described  peculiar  organisms 
in  the  blood  of  healthy  rats  in  India.  When  first  noticed 

*  Abstract  of  paper  by  the  Author,  Journ.  Roy.  Micros.  Soc., 
read  November  loth,  1886.  See  also  papers  on  the  Micro-parasites 
of  Malaria  by  Laveran,  Marchiafava  and  Celli. 

J  Lewis,  Microscopic  Organisms  in  the  Blood  of  Man  and 
Animals.  Calcutta,  1879  (with  photographs) ;  and  Quart.  Journ. 
Micr.  Sci.>  Ixxiii.  (1879),  pp.  109-14,  and  xxiv.  (1884),  pp.  357-69. 


APPENDIX.  351 

they  were  thought  to  be  vibrios  or  spirilla.  A  drop  of 
blood  under  examination  appeared  to  quiver  with  life,  and 
on  diluting  the  blood,  motile  filaments  could  be  seen 
rushing  through  the  serum,  and  tossing  the  blood- 
corpuscles  about  in  all  directions.  Under  careful  exami- 
nation the  filaments  were  found  to  consist  of  a  thicker 
portion  or  body,  with  at  one  end  a  flagellum  (Fig.  129). 
After  fixing  with  osmic  acid  they  measured  O'8 — I  JLL  in 
width,  and  20 — 30  JJL  in  length  ;  the  flagellum  was  about 
as  long  as  the  body,  so  that  the  total  length  of  the 
organism  was  about '50  JA.  Lewis  detected  these  parasites 
in  29  per  cent,  of  the  species  Mus  decumanus  and  Mus 


FIG.  129. — PARASITES  IN  THE  BLOOD  OF  RATS  [after  Lewis]. 

rufescens.  Though  they  had  many  features  in  common 
with  motile  organisms  of  vegetable  origin,  they  appeared 
to  approach  much  more  closely  to  the  Protozoa,  more  par- 
ticularly several  of  the  species  of  Dujardin's  Cercomonas. 

Wittich*  discovered,  in  the  blood  of  hamsters,  whip- 
like  bodies  with  lively  movements.  They  resembled 
frog's  spermatozoa,  possessing  a  thick  portion  continued 
into  a  long  lash-like  thread.  Wittich  considered  them 
identical  with  the  organisms  described  by  Lewis,  and  they 
also  were  observed  in  apparently  healthy  animals.  Kochf 
later  met  with  the  same  organisms. 

*  "  Spirillen  im  Blut  von  Hamstern,"  Centralbl.  f.  Med.  Wiss. 
1881,  No.  4. 
t  Mittheilungen  aus  dem  Kaiser  lick.  Gesundh.  Amt.  1881.     .8. 


352  APPENDIX. 

Flagellated  organisms  in  the  blood  of 
horses,  mules,  and  camels. — In  India  a  fatal 
disease,  known  by  the  natives  as  Surra,  occurs  in 
horses,  mules,  and  camels.  The  malady  is  described 
as  a  blood  disease,  characterised  by  fever,  accompanied 
by  jaundice,  petechiae  of  mucous  membranes,  great 
prostration,  and  rapid  wasting1,  terminating  in  death. 
Evans*  observed  the  presence  of  a  parasite  in  the  blood, 
and  by  means  of  subcutaneous  inoculation,  and  by  the 
introduction  into  the  stomach  of  blood  containing  the 
parasites,  the  disease  was  transmitted  to  healthy  animals. 

Steel,f  who  was  deputed  to  investigate  this  disease  in 
British  Burma,  also  found  the  parasite  in  all  cases,  and 
further  observed  that  it  appeared  as  the  temperature  rose 
and  disappeared  during  the  apyrexial  periods.  This 
observer  concluded  that  the  organism  was  a  spiral  bac- 
terium, and  named  it,  after  its  discoverer,  Spirochceta 
Evansi. 

Flagellated  organisms  in  the  blood  of  fish. 
— In  the  blood  of  mud-fish  (Cobitis  fossilis]  MitrophanowJ 
observed  the  presence  of  peculiar  micro-parasites  (Fig. 
130).  As  a  i  per  cent,  salt  solution  had  been  added  to 
the  blood  under  examination,  it  occurred  to  Mitrophanow 
that  they  were  possibly  the  cytozoa  described  by  Gaule  ; 
but  this  idea  was  dismissed  by  the  fact  that  they  were 
found  in  blood  to  which  no  salt  solution  was  added. 
Their  size  varied  from  30  to  40  p,  in  length,  and  i  to  I  *5  /A 
in  width.  At  first  their  rapid  movements  baffled  examina- 
tion, but  as  the  rapidity  lessened  there  was  the  appearance 
of  a  curling  movement  in  the  body  portion,  and  a  swinging 
movement  of  the  lash.  The  organism  moved  in  the 
direction  of  the  lash,  the  anterior  end  of  the  body  being 

*  Evans,  Report  published  by  the  Punjab  Government  Military 
Department,  No.  439.  1880. 

t  Steel,  A.V.D.,  An  Investigation  into  an  Obscure  and  Fata 
Disease  among  Transport  Mules  in  British  Burma.     1885. 

t  "  Beitrage  zur  Kenntniss  der  Hamatozoen,"  BioL  Centrabl., 
iii.,  1883,  pp.  35-44. 


APPENDIX. 


353 


more  pointed  than  the  posterior,  and  gradually  fining  off 
into  the  lash.  When  the  body  seemed  to  rest,  the  lash 
might  be  seen  to  whip  out  in  all  directions.  As  the 
movement  of  the  body  gradually  diminished,  it  appeared 
to  have  a  complicated  screw-form,  the  axis  of  the  screw 
corresponding  to  the  body,  to  which  an  undulating  mem- 
brane is  fastened  spirally.  This  could  be  distinguished 
when  the  organism  was  dying,  because  the  body  in  death 
contracted,  and  the  membrane  then  looked  like  a  spiral 


FlG.    130.— H^MATOMONAS   COBITIS. 

fft  First  variety  ;  b,  second  variety ;  c,  third  variety. 

d,  First  variety  in  a  state  of  diminished  activity. 

e,  The  same  after  treatment  with  osmic  acid. 

[After  Mitrophanovv.j 

addition.      Thus    the   organism    consisted    of  a   body,    a 
spiral  membrane,  and  a  flagellum. 

With  higher  magnification  the  organism  appeared  to 
consist  of  a  refractive,  strongly-contractile,  protoplasmic 
substance,  which,  when  death  occurred,  formed  a  shape- 
less mass.  In  the  same  blood  two  other  forms  were 
observed,  one  without  a  membrane,  but  having  two  highly- 

23 


354  APPENDIX. 

refractive  spherules  in  the  protoplasm,  and  another  with 
neither  membrane  nor  flagellum,  consisting  of  very  granular 
protoplasm  with  several  refractive  spherules,  and  capable 
of  protruding  processes  like  pseudopodia. 

In  the  blood  of  the  German  carp  (Cyprinus  Carassius) 
Mitrophanow  describes  a  parasite  which  is  perceptibly 
larger,  and  possesses  an  undulating  membrane  fastened 
along  the  edge  of  the  long  body  (Fig.  131).  When  the 
body  bent  first  towards  one  side  and  then  to  the  other,  a 


FIG.  131. — ORGANISMS  IN  THE  .BLOOD  OF  THE  CARP. 

a,  6,  c,  hcematomonas  carassii.    </,  e,f,  g,  h,  Other  organisms  in  the  same 
blood  [after  Mitrophanow]. 

wave-like  movement  was  observable   at   the  free   edge  of 
this  membrane. 

Jn  Cobitis  fossilis  these  parasites  were  found  in  all  the  fish 
examined  except  one,  and  in  greater  numbers  in  the  hot 
months.  In  Cyprinus  Carassius  they  were  only  found  occa- 
sionally. Mitrophanow  described  other  varieties,  which  he 
considered  were  possibly  not  complete  organisms,  but  deve- 
lopmental forms.  He  considered  that  these  organisms  were 


APPENDIX.  355 

infusoria  between  the  genera  Cercomonas  and  Trichomonas, 
with  great  similarity  to  the  Trichomonas  described  in  the 
Lieberkiihn's  glands  of  fowls  and  ducks  (Eberth*). 

On  account  of  their  special  habitat  Mitrophanow 
suggested  a  new  genus  (Hcematomonas)y  defining  this  genus 
as  follows  : — Parasites  of  normal  fish-blood,  worm-like, 
actively-moving  organisms,  with  indistinct  differentiation 
of  body  parenchyma.  Bodies  pointed  at  both  ends,  30 
to  40  fji  long  and  I  to  1*5  jit  wide.  May  possess  in  front 
a  flagellum,  and  on  one  side  an  undulating  membrane. 

Species : — 

Hcematomonas  cobitis. — Body  provided  with  a  spiral 
membrane  and  a  flagellum  at  the  fore-end.  Parenchyma 
of  body  homogeneous.  Second  variety,  body  and  flagel- 
lum only.  Movement  undulatory,  body  containing  highly 
refractive  spherules.  Third  variety,  plasma-like  body, 
without  membrane  or  flagellum  ;  quickly  changes  form  by 
sending  out  processes  laterally,  and  contains  two  to  four 
refractive  spherules.  Blood  of  •  Cobitis  fossilis. 

Hcematowonas  carassii. — Long  bodies,  with  narrow 
membrane  attached  along  the  whole  length  ;  less  actively 
motile.  Several  forms  also  observed  strikingly  smaller 
than  the  above  ;  many  disc-shaped.  Often  seen  attached 
to  a  red  corpuscle,  setting  them  in  motion  by  their  move- 
ments. Blood  of  Cyprinus  carassius. 

Quite  recently  the  author  has  investigated  the  parasites 
found  in  the  disease  known  as  Surra,  and  came  to  the 
following  conclusions  : 

In  stained  preparations  the  somewhat  tapering  central 
portion,  or  body,  of  the  parasite  is  found  to  be  continuous 
at  one  end  with  a  whip-like  lash,  and  at  the  other  end  to 
terminate  in  an  acutely-pointed  stiff  filament,  or  spine-like 
process.  Here  and  there,  possibly  from  injury  or  want  of 
development,  the  spine-like  process  appears  to  be  blunted 

*  Vide  Leuckart,   The  Parasites  of  Man,  translated  by  Hoyle, 
p.  248. 


356  APPENDIX. 

or  absent.  By  very  careful  focusing  on  the  upper  edge  of 
the  central  portion,  the  author  discovered  the  existence, 
much  more  markedly  in  some  of  the  parasites  than  in 
others,  of  a  longitudinal  membrane  with  either  a  straight 
or  undulating  margin  (Fig.  132).  The  membrane  is 
attached  along  the  body,  arising  from  the  base  of  the 
rigid  filament,  and  becomes  directly  continuous  at  the 
opposite  end  with  the  flagellum.  In  some  cases  the  edge 
only  is  deeply  stained,  giving  the  appearance  of  a  thread 
continuous  with  the  flagellum,  so  that  one  might  be  easily 
led  to  overlook  the  membrane,  and  imagine  that  the 


FIG.  132. — "SURRA"  PARASITES  OCCURRING  SINGLY  AND  FUSED. 
From  preparations  stained  with  magenta,  X  1200. 

flagellum  arose  from  the  opposite  end  of  the  body,  at  the 
base  of  the  spine-like  process. 

Close  to  the  base  of  the  spine-like  process  a  clear 
unstained  spot  is,  in  many  parasites,  easily  distinguished, 
and  at  the  opposite  end  there  is,  in  some,  the  appearance 
of  the  deeply-stained  protoplasmic  contents  having  con- 
tracted within  the  faintly-stained  cell-wall.  Where  the 
longitudinal  membrane  has  a  wavy  outline  the  undulations 
are  much  more  marked  in  some  cases  than  in  others. 
Here  and  there  the  wavy  outline  appears  first  on  one  side 
of  the  central  portion,  and  then  on  the  other,  but  there 
never  is  any  waving  outline  on  both  sides  of  the  same 
part  of  the  body,  and  this  is  explained  by  careful  exami- 


APPENDIX.  357 

nation,  which  shows  that  in  dying  the  somewhat  ribbon- 
like  parasite  has  become  doubled  on  itself. 

Owing  to  the  somewhat  curved  and  twisted  shape  of 
the  parasite  and  the  curling  of  the  flagellum,  in  the  stained 
preparations,  it  was  difficult  to  make  exact  measurements  ; 
but  the  average  width,  according  to  whether  the  membrane 
was  visible  or  not,  varied  from  I  to  2  /*,  and  the  length 
of  the  body  from  20  to  30  p.  The  flagellum  was  about 
the  same  length  as  the  body. 

From  these  observations  (especially  the  discovery  of  the 
undulating  longitudinal  membrane)  the  author  recognised 
a  very  close  resemblance  to  Mitrophanow's  descriptions, 
and  concluded  that,  if  we  followed  the  classification 
adopted  by  Mitrophanow,  the  genus  Hcematomonas  must 


FIG.  133. — A  MONAD  IN  RAT'S  BLOOD,  X  3000. 

The  organism  is  represented  at  partial  rest,  with  its  posterior  filament  im 
pinging  on  a  corpuscle,  and  showing  the  undulating  longitudinal  membrane, 
the  long  flagellum,  and  the  refractive  spherules  in  the  granular  protoplasm. 

not  be  restricted  to  organisms  in  fish-blood.  It  must  be 
expanded  to  include  this  parasite  of  mammalian  blood, 
which  should  in  that  case  be  named  H&matomonas  Evansi, 
rather  than  Spirochceta  Evansi,  as  proposed  by  Steel. 

In  the  course  of  this  investigation  the  author  was  led 
to  examine  the  blood  of  rats  obtainable  in  this  country. 
Organisms  were  discovered  in  the  blood  of  about  25 
per  cent,  of  common  brown  rats  ;  and,  after  examining 
them  with  various  objectives,  from  a  \  dry  to  a  ^  oil- 
immersion  of  Powell  and  Lealand,  the  following  con- 
clusions were  arrived  at  : — That  they  are  polymorphic, 
presenting  for  the  most  part  slightly  tapering  bodies 


358  APPENDIX. 

which  terminate  at  one  end  in  a  stiff,  immotile,  acutely- 
pointed,  flexible  filament  or  spine-like  process,  and  at  the 
opposite  end  are  provided  with  a  long  flagellum,  while 
longitudinally  attached  a  delicate  undulating,  fin-like 
membrane  can  be  traced,  which  starts  from  the  base  of 
the  posterior  filament,  and  becomes  directly  continuous 
with  the  flagellum  (Fig.  133). 

With  careful  illumination  the  body  is  found  to  be 
distinctly  granular,  with  one  or  more  highly-refractive 
spherules.  When  the  rapid  movement  is  arrested  the 
undulating  membrane  is  distinctly  visible.  The  best 
opportunity  occurs  for  seeing  this  when  the  organism 


FIG.  134.— MONADS  IN  RAT'S  BLOOD,  x  1200. 

a,  A  monad  threading  its  way  among  the  blood-corpuscles  ;  b.  another  with 
pendulum  movement  attached  to  a  corpuscle  ;  c,  angular  forms ;  d,  encysted 
forms;  e  and/,  the  same  seen  edgeways. 

comes  to  partial  rest  with  its  stiff  filament  against  a 
corpuscle,  as  if  to  obtain  a  point  d'apflui,  while  lashing 
its  flagellum  in  all  directions  (Fig.  134,  £).  At  other 
times,  when  the  parasite  has  impinged  with  its  posterior 
extremity  against  a  corpuscle,  or  the  stiff  filament  is 
apparently  entangled  in  debris,  the  movements  of  the 
organism  give  one  the  idea  of  its  endeavouring  to  set 
itself  free. 

In  the  active  state  the  thicker  portion,  or  body,  appears 


APPENDIX.  359 

to  twist  and  bend  from  side  to  side  with  great  activity. 
The  organism  can  turn  completely  round  with  lightning 
rapidity,  so  that  the  flagellum,  at  one  moment  lashing 
in  one  direction,  is  suddenly  observed  working  in  the 
opposite  direction.  Then  suddenly  the  organism  makes 
progression,  and  it  can  be  distinctly  seen  to  move  in  the 
direction  of  tJie  flagellant,  the  flagellum  threading  its  way 
between  tJu  corpuscles  and  drawing  the  rest  of  the  organism 
after  it.  By  treating  cover-glass  preparations  with  osmic 
acid,  the  appearances  corresponded  exactly  with  photo- 
graphs of  the  organisms  observed  by  Lewis  in  India,  so 
that  the  author  has  no  doubt  of  their  identity,  in  spite  of 
the  descriptions  not  completely  according.  A  great  like- 
ness to  the  organisms  described  by  Mitrophanow,  and  to 
the  Surra  parasite,  as  just  described,  was  obvious  ;  and 
after  staining  the  rat  parasites  the  closest  examination 
confirmed  the  belief  that  they  were  morphologically 
identical  with  the  stained  parasites  of  Surra. 

The  cover-glasses  with  a  thin  layer  of  blood  should  be 
passed  through  the  flame  of  a  Bunsen  burner  in  the  way 
commonly  employed  for  examining  micro-organisms,  and 
stained  with  an  aqueous  solution  of  fuchsine,  methyl-violet, 
or  Bismarck  brown.  The  following  method  will,  how- 
ever, be  found  most  instructive.  Use  freshly-prepared 
saturated  solution  of  fuchsine  or  methyl-violet  in  absolute 
alcohol,  and  put  a  drop  with  a  pipette  on  the  centre  of  the 
preparation  ;  do  not  disturb  the  drop-form  for  a  few 
moments  ;  then,  before  the  alcohol  has  evaporated,  wash 
off  the  excess  of  stain.  It  will  be  found  that  where  the 
drop  rested  the  organisms  will  be  very  deeply  stained, 
while  in  the  surrounding  area  the  colour  will  vary  in 
intensity. 

By  the  effect  of  the  different  degrees  of  staining  much> 
may  be  learnt  (Fig.  135).  In  one  organism  the  body  and 
entire  membrane  will  be  equally  stained  ;  in  another  the 
margin  of  the  membrane  only.  In  some  the  posterior 
stiff  filament  is  stained,  and  at  its  base  a  darkly-stained 


360  APPENDIX. 

speck  is  very  striking  ;  and  in  other  cases  again  the 
posterior  filament  is  only  faintly  tinged,  or  an  unstained 
spot  occurs  near  its  base. 

The  morphological  identity  of  the  rat  and  Surra 
parasites  is  thus  established,  and  both  seem  morpho- 
logically identical  with  the  organism  of  Mitrophanow. 
If  we  follow  Mitrophanow  we  must,  therefore,  enlarge 
his  genus  of  Hcematomonas.  The  author  ventures,  how- 
ever, to  disagree  with  Mitrophanow  in  the  advisability  of 
adopting  this  entirely  new  generic  name.  Mitrophanow 
suggested  this  new  term  because  of  the  special  habitat 
—  normal  fish-  blood  —  of  the  species  he  discovered.  But 


FIG.  135.  —  MONADS  IN  RAT'S  BLOOD,  showing  membrane  under  different 
aspects,  blood-corpuscles  some  crenated,  and  stained  discs,  X  I2CO. 

the  characteristic  features  of  these  organisms  are  the 
characteristic  marks  of  the  genus  Trichomonas*  They 
are,  therefore,  embraced  by  the  genus  Trichomonas,  and 
there  is  no  need  to  create  a  new  one.  If  it  were  not 
for  the  different  description  given  by  Mitrophanow  of  the 
organism  in  the  blood  of  Colitis  fossilis,  the  author  would 
be  inclined  to  say  that  all  these  organisms  belonged  to 
one  and  the  same  species,  which  might  well  be  named 
Trichomonas  sanguinis.  The  monads  in  the  rat  and  the 
Surra  parasite  have  been  shown  to  be  morphologically 
identical  with  each  other,  and  both,  as  far  as  one  can 
*  Vide  Leuckart,  The  Parasites  of  Man,  translated  by  Hoyle,  1886. 


APPENDIX.  361 

judge  from  the  description,  are  morphologically  identical 
with  the  monad  in  the  blood  of  the  carp.  We  have,  how- 
ever, seen  that  the  organism  in  Surra  is  believed  to  be 
pathogenic,  and  too  much  stress  must  not  be  laid  on 
morphological  identity.  There  is  strong  evidence  in 
.favour  of  believing  in  its  pathogenic  properties  ;  but,  at 
the  same  time,  it  must  be  borne  in  mind  that  the  organism 
has  never  been  isolated  apart  from  the  blood,  and  the 
disease  then  produced  by  its  introduction  into  healthy 
animals.  It  is  quite  possible  that  the  parasites  in  Surra 
are  only  associated  with  the  disease,  the  impoverished 
blood  affording  a  suitable  nidus  for  their  development, 
while  the  contaminated  water  may  be  the  common  source 
of  the  organism  and  of  the  disease.  On  the  other  hand, 
the  organism  in  the  rat  is  found  in  apparently  perfectly 
healthy,  well-nourished  animals. 


APPENDIX    C. 
EXAMINATION     OF    AIR. 

THE  air,  as  is  well  known,  contains  in  suspension  mineral, 
animal,  and  vegetable  substances.  The  mineral  world  is 
represented  by  such  substances  as  silica,  silicate  of  alu- 
minium, carbonate  and  phosphate  of  calcium,  which  may 
be  raised  from  the  soil  by  the  wind,  and  particles  of 
carbon,  etc.,  which  gain  access  from  accidental  sources. 
Belonging  to  the  animal  kingdom  we  find  the  debris  of 
perished  creatures  as  well  as  sometimes  living  animals. 
The  vegetable  world  supplies  micrococci,  bacilli,  and  other 
forms  of  the  great  family  of  bacteria,  spores  of  other 
fungi,  pollen  seeds,  parts  of  flowers,  and  so  forth.  The 
air  of  hospitals  and  sick  rooms  has  been  found  to  be 
especially  rich  in  vegetable  forms,  e.g.,  fungi  and  spores 
have  been  observed  as  present  in  particularly  large  numbers 
in  cholera  wards,  spores  of  Tricophyton  have  been  dis- 
covered in  the  air  of  hospitals  for  diseases  of  the  skin,  and 
achorion  in  wards  with  cases  of  favus.  The  tubercle-bacil- 


362  APPENDIX. 

lus  is  said  to  have  been  detected  in  the  breath  of  patients 
suffering  from  phthisis. 

These  points  indicate  that,  in  addition  to  the  interest 
for  the  microbiologist,  considerable  importance  from  a 
hygienic  point  of  view  must  be  attached  to  the  sys- 
tematic examination  of  the  air.  Especially  a  knowledge 
of  the  microbes  which  are  found  in  the  air  of  marshy  and 
other  unhealthy  districts,  and  in  the  air  of  towns,  dwel- 
lings, hospitals,  workshops,  factories,  and  mines,  will  be 
of  practical  value. 

Miquel,*  who  has  particularly  studied  the  bacteria  in  the 
air,  has  found  that  their  number  varies  considerably.  The 
average  number  per  cubic  metre  of  air  for  the  autumn 
quarter  at  Montsouris  is  given  as  142,  winter  quarter  49, 
spring  quarter  85,  and  summer  quarter  105.  In  air  col- 
lected 2,000  to  4,000  metres  above  the  sea-level,  not  a 
single  bacterium  or  fungus  spore  was  furnished,  while  in 
10  cubic  metres  of  air  from  the  Rue  de  Rivoli  (Paris) 
the  number  was  computed  at  55,000. 

The  simplest  method  for  examining  the  organisms  in  air 
consists  in  exposing  plates  of  glass  or  microscopic  slides 
coated  with  glycerine,  or  a  mixture  of  glycerine  and  glucose 
which  is  stable,  colourless,  and  transparent.  Nutrient 
gelatine  spread  out  on  glass  plates  (p.  81)  may  be  exposed 
to  the  air  for  a  certain  time,  and  then  put  aside  in  damp 
chambers  for  the  colonies  to  develop.  Sterilised  potatoes 
prepared  in  the  usual  way  (p.  82)  may  be  similarly  exposed. 
In  both  the  last  mentioned  methods  separate  colonies 
develop,  which  may  be  isolated  as  already  described,  and 
pure  cultivations  carried  on  in  various  other  nutrient  media 
(p.  79).  Nutrient  gelatine  has  also  been  employed  in  the 
special  methods  of  Koch  and  Hesse. 

Koch's   Apparatus. — This  consists  of  a   glass  jar 

about  six  inches  high,  the  neck  of  which  is  plugged  with 

cotton  wool.      In  the  interior  is  a  shallow  glass  capsule, 

which  can  be  removed  by  means  of  a  brass   lifter.     The 

*  Miquel,  Grganismes  vivants  de  V atmosphere. 


APPENDIX.  363 

whole  is  sterilised  by  exposure  to  150°  C.  for  an  hour  in 
the  hot-air  steriliser.  The  nutrient  gelatine  in  a  stock- 
tube  is  liquefied,  and  the  contents  emptied  into  the  glass 
capsule.  The  jar  is  exposed  to  the  air  to  be  examined 
for  a  definite  time,  the  cotton  wool  plug  replaced,  and 
the  apparatus  set  aside  for  the  colonies  to  develop. 

Hesse's    Apparatus  (Fig.  136).— The  advantage  of 


FIG.  136.— HESSE'S  APPARATUS. 

this  apparatus  consists  in  that  a  known  volume  of  air  can 
be  examined.  A  glass  cylinder,  70  cm.  long  and  3-5  cm. 
in  diameter,  is  closed  at  one  end  by  an  india-rubber  cap, 
perforated  in  the  centre.  Over  this  fits  another  cap,  which 
is  not  perforated.  The  opposite  end  of  the  cylinder  is 
closed  with  a  caoutchouc  stopper,  perforated  to  admit 
a  glass  tube  plugged  with  cotton  wool.  The  tube  can  be 
connected  by  means  of  india-rubber  tubing  with  an 


j  64  APPENDIX. 

aspirating  apparatus.  This  apparatus  consists  of  a  couple 
of  litre-flasks,  suspended  by  hooks  from  the  tripod  stand 
which  supports  the  whole  apparatus.  The  cylinder,  caps, 
and  plug,  are  washed  with  solution  of  corrosive  sub- 
limate, and  then  with  alcohol.  After  being  thus  cleansed, 
50  ccm.  of  nutrient  gelatine  are  introduced,  and  the  whole 
sterilised  by  steaming  for  half  an  hour  for  three  successive 
days.  After  the  final  sterilisation  the  cylinder  is  rotated 
on  its  long  axis,  so  that  the  nutrient  medium  solidifies 
in  the  form  of  a  coating  over  the  whole  of  the  interior. 
When  required  for  use,  the  cotton-wool  plug  is  removed 
from  the  small  glass  tube,  and  the  latter  connected  with 
the  upper  flask  by  means  of  the  india-rubber  tubing. 

The  apparatus  is  placed  in  the  air  which  is  to  be 
examined,  the  outer  india-rubber  cap  removed  from 
the  glass  cylinder,  and  the  upper  flask  tilted  until  the 
water  begins  to  flow  into  the  lower  one.  The  emptying 
continues  by  syphon  action,  and  air  is  drawn  in  along 
the  cylinder  to  replace  the  water.  When  the  upper  flask 
is  empty,  the  position  of  the  two  is  reversed,  and  the  flow 
again  started.  When  a  sufficient  volume  has  been  drawn 
through  the  cylinder  the  outer  cap  and  the  cotton-wool 
plug  are  replaced,  and  it  is  set  aside  for  the  colonies  to 
develop.  As  an  example,  twenty-five  litres  of  air  from 
an  open  square  in  Berlin  gave  rise  to  three  colonies  of 
bacteria  and  sixteen  moulds ;  on  the  other  hand,  two 
litres  from  a  school-room  just  vacated  by  the  scholars 
gave  thirty-seven  colonies  of  bacteria  and  thirty-three 
moulds. 

Various  forms  of  "  aeroscopes  "  and  "  aeroniscopes  "  have 
from  time  to  time  been  employed.  Pouchet's  aeroscope 
consists  of  a  small  funnel,  drawn  out  to  a  point  below 
which  is  a  glass  slip  coated  with  glycerine.  The  end  of 
the  funnel  and  the  glass  slip  are  enclosed  in  an  air-tight 
chamber,  from  which  a  small  glass  tube  passes  out  con- 
nected by  india-rubber  tubing  with  an  aspirator.  The 
air  passing  down  the  funnel  strikes  upon  the  glycerine, 


APPENDIX.  365 

which  arrests  any  solid  particles.  For  a  description  of 
the  more  exact  apparatus  employed  by  Maddox,  Cun- 
ningham, and  Miquel  reference  should  be  made  to  the 
writings  of  these  authors,  and  particularly  to  the  treatise 
published  by  the  last-named. 


APPENDIX   D. 

EXAMINATION    OF    SOIL. 

SURFACE-SOIL,  or  mould,  is  exceedingly  rich  in  bac- 
teria. Miquel,  e.g.)  has  computed  that  there  exists  in  a 
gramme  of  soil  an  average  of  750,000  germs  at  Mont- 
souris,  1,300,000  in  the  Rue  de  Rennes,  and  2,100,000 
in  the  Rue  de  Monge.  As  agents  of  putrefaction  and 
fermentation  they  play  a  very  important  role  in  the 
economy  of  nature,  but  there  exist  in  addition  bacteria 
in  the  soil  which  are  pathogenic  in  character.  Pasteur 
has  succeeded  in  isolating  from  the  earth  the  bacillus  of 
anthrax,  and  sheep,  sojourning  upon  a  plot  of  ground 
where  animals  which  have  died  of  anthrax  had  been 
buried,  succumbed  to  the  disease.  Pasteur  considered 
that  the  spores  were  conveyed  by  worms  from  buried 
beasts  to  the  surface  soil.  The  bacillus  of  malignant 
oedema  is  also  present  in  soil,  and  Nicolaier  has  culti- 
vated a  bacillus  from  earth  which  produced  tetanus  in 
mice,  rabbits,  guinea-pigs,  and  other  animals. 

To  obtain  a  cultivation  of  the  microbes  in  soil  a 
sample  of  the  latter  must  be  first  dried  and  then 
triturated.  It  may  then  be  shaken  up  with  distilled 
water,  and  from  this  a  drop  transferred  to  sterilised 
bouillon.  The  employment  of  solid  media  is,  however, 
much  more  satisfactory  :  A  sample  of  earth  is  collected, 
dried,  and  triturated,  and  a  small  quantity  sprinkled  over 
the  surface  of  nutrient  gelatine  prepared  for  a  plate-culti- 
vation. In  another  method  the  gelatine  is  liquefied  in  a 


366  APPENDIX. 

test-tube,  the  powder  added,  and,  in  the  usual  way,  dis- 
tributed throughout  the  medium,  which  is  then  poured 
out  upon  a  glass  plate.  Just  in  the  same  way  the 
dust  which  settles  from  the  air  in  houses  and  hospitals, 
or  food  substances  in  powder,  may  be  distributed  over 
nutrient  gelatine,  and  the  micro-organisms  which  develop 
studied,  both  as  to  their  morphological  and  biological 
characteristics. 


APPENDIX  E. 

EXAMINATION    OF    WATER. 

As  in  the  case  of  air,  so,  too,  in  that  of  water  a  knowledge 
of  the  micro-organisms  which  may  be  present  is  not  only 
of  interest  to  the  microbiologist,  but  of  the  greatest 
importance  in  practical  hygiene.  Common  putrefactive 
bacteria  and  vibrios  may  not  be  hurtful  in  themselves,  but 
they  indicate  the  probability  of  the  presence  of  organic 
matter  in  some  of  which  there  may  be  danger.* 

The  Microzyme  Test,  which  was  introduced  for 
their  detection,  consisted  in  adding  three  or  four  drops  of 
the  sample  of  water  to  I  or  2  ccm.  of  Pasteur's  fluid, 
the  nourishing  fluid  having  been  previously  boiled  in  a 
sterilised  test-tube.  If  the  microzymes  or  their  germs 
existed  in  the  water,  the  liquid  in  a  few  days  became 
milky  from  the  presence  of  countless  bacteria.  This 
test  is  of  no  real  value,  for  it  does  little  more  than  indi- 
cate that  bacteria  were  present,  which  we  may  accept  as 
being  present  in  ice  and  all  ordinary  water.  On  the  other 
hand,  the  bacteriological  test  of  Professor  Koch  is  a  most 
valuable  addition  to  the  usual  methods  of  water-analysis. 
It  enables  us  not  only  to  detect  the  presence  of  bacteria, 
but  to  ascertain  approximately  their  number,  and  to  study 
very  minutely  their  morphological  and  biological  charac- 

*  Parkes,  Manual  of  Practical  Hygiene.     1 883 . 


APPENDIX,  367 

teristics.  The  importance  of  a  thorough  acquaintance 
with  the  life-history  of  the  individual  micro-organisms 
cannot  be  too  strongly  insisted  upon.  For  example,  by 
such  means  the  spirillum  of  Asiatic  cholera  can  be  dis- 
tinguished from  other  comma-shaped  organisms,  and 
inasmuch  as  its  presence  may  be  an  indication  of  con- 
tamination with  choleraic  discharges,  such  water  should 
be  condemned  for  drinking  purposes,  even  though  we  may 
not  yet  be  in  a  position  to  affirm  that  the  microbe  is  the 
cause  of  the  disease.  The  test,  in  short,  consists  in  making 
plate-cultivations  of  a  known  volume  of  water,  counting 
the  colonies  which  develop,  isolating  the  micro-organisms, 
and  studying  the  characters  of  each  individual  form. 

Collection  and  Transport  of  Water  Samples. 
— Erlenmeyer's  conical  flasks  of  about  100  ccm.  capacity 
may  be  employed  with  advantage  for  collecting  the 
samples  of  water.  They  are  cleansed,  plugged,  and 
sterilised  in  the  hot-air  steriliser.  When  required  for  use, 
the  plug  is  removed  and  held  between  the  ringers,  which 
must  not  touch  the  part  which  enters  the  neck  of  the 
flask.  About  30  ccm.  of  the  water  to  be  examined  are 
introduced  into  the  flask,  and  the  plug  must  be  quickly 
replaced  and  covered  with  a  caoutchouc  cap.  If  collected 
from  a  tap,  the  water  should  first  be  allowed  to  run  for  a 
few  minutes,  and  the  sample  should  be  received  into  the 
flask  without  the  neck  coming  into  contact  with  the 
tap.  From  a  reservoir  or  stream  the  flasks  may  be  filled 
by  employing  a  sterilised  pipette.  During  transport  con- 
tact between  the  water  and  cotton-wool  plug  must  be 
avoided,  and  if  likely  to  occur  the  sample  must  be  collected 
and  forwarded  in  a  Sternberg's  bulb  (p.  31). 

Examination  by  Plate  Cultivation. — The 
apparatus  for  plate-cultivation  should  be  arranged  as 
already  described.  Crushed  ice  may  be  added  to  the 
water  in  the  glass  dish  to  expedite  the  setting  of  the 
gelatine,  so  that  the  plate  may  be  transferred  as  quickly 
as  possible  to  the  damp- chamber.  The  caoutchouc  cap 


368  APPENDIX. 

is  removed  from  the  flask,  and  the  cotton-wool  plug 
singed  in  the  flame  to  prevent  contamination  from 
adventitious  germs  on  the  outside  of  the  plug.  The 
flask  is  then  held  slantingly  in  the  hand,  and  the  plug 
twisted  out  and  retained  between  the  fingers.  With  a 
graduated  pipette  a  drop  of  the  sample  is  transferred  to  a 
tube  of  liquefied  nutrient  gelatine,  and  the  plug  of  the 
flask  and  tube  quickly  replaced.  If  the  water  is  very 
impure,  it  may  be  necessary  to  first  dilute  the  sample 
with  sterilised  water.  The  inoculated  tube  must  be 
gently  inclined  backwards  and  forwards  and  rolled  as 
already  explained,  to  distribute  the  germs  throughout  the 
gelatine  (p.  75),  and  the  gelatine  finally  poured  on  a  plate. 


FIG.  137,— APPARATUS  FOR  ESTIMATING  THE  NUMBER  OF  COLONIES  IN 
A  PLATE-CULTIVATION. 

When  the  gelatine  has  set,  the  plate  is  transferred  to  a 
damp  chamber,  which  should  be  carefully  labelled  and  set 
aside  in  a  place  of  moderate  temperature.  In  about  two 
or  three  days  the  cultivation  may  be  examined.  In  some 
cases  the  colonies  may  be  counted  at  once  ;  more  frequently 
they  are  so  numerous  that  the  plate  must  be  placed 
on  a  dark  background,  and  a  special  process  resorted 
to.  A  glass-plate,  ruled  by  horizontal  and  vertical  lines 
into  centimetre  squares,  some  of  which  are  again  sub- 
divided into  ninths,  is  so  arranged  on  a  wooden  frame  that 
it  can  cover  the  nutrient  gelatine-plate  without  touching 
it  (Fig.  137).  A  lens  is  added  to  assist  in  discovering 
minute  colonies.  If  then  the  colonies  are  very  numerous, 


APPENDIX.  369 

the  number  in  some  small  divisions  is  counted,  if  less  in 
some  large  ones,  and  an  average  is  obtained  from  which 
the  number  of  colonies  on  the  entire  surface  is  calculated. 
A  separate  calculation  of  the  liquefying  colonies  should 
be  also  made,  and  their  number,  as  well  as  the  total 
number  of  colonies  present  in  I  ccm.  of  the  sample,  re- 
corded. Any  peculiar  macroscopical  appearances,  colour, 
etc.,  should  be  noted,  and  then  the  microscopical  appear- 
ances of  the  colonies  studied.  Lastly,  examination  of 
the  individual  organisms  should  be  made  by  cover-glass- 
preparations,  and  by  inoculation  of  nutrient  gelatine,  pota- 
toes, and  other  media. 

Examination  by  Test-Tube  Cultivation. — A 
drop  of  the  sample  of  water  may  also  be  added  to  liquefied 
nutrient  gelatine  in  a  tube,  the  organisms  distributed  as 
already  explained  (p.  75,)  and  the  gelatine  allowed  to 
solidify  in  the  tube.  A  rough  comparison  of  water 
samples  may  be  made  in  this  way. 

Microscopic  Examination. — A  drop  of  the 
water  may  be  mounted  and  examined  in  the  way 
described  under  drop-cultivations  (p.  94),  or  a  drop  is 
allowed  to  evaporate  on  a  cover-glass  placed  under  a 
bell-glass.  This  is  then  passed  three  times  through  the 
flame,  and  stained  in  the  usual  manner.  The  examina- 
tion of  rain  water,  drinking  water,  tap  water,  sea  water, 
various  liquids  and  infusions,  etc.,  by  these  methods  opens 
up  a  wide  field  for  research.  Pettenkofer  has  shown  that 
impregnation  with  carbonic  acid  of  water  containing  many 
bacteria  diminishes  the  number  of  the  latter.  The  ex- 
amination of  waters  before  and  after  filtration,  or  after 
addition  of  chemical  substances,  are  matters  which  require 
further  investigation. 


APPENDIX    F. 

CHRONOLOGICAL    BIBLIOGRAPHY. 


A.  METHODS. 

B.  MORPHOLOGY  AND   CLASSIFICATION. 

C.  GENERAL   BIOLOGY. 

D.  ZYMOGENIC    SAPROPHYTES    AND    FERMENTATION. 

E.  CHROMOGENIC   SAPROPHYTES 

F.  SIMPLE   SAPROPHYTES. 

G.  PTOMAINES    AND    PUTREFACTION. 
H.    ANTISEPTICS    AND    DISINFECTANTS. 

I.    IMMUNITY. 

J.    BACTERIA  ASSOCIATED  WITH   DISEASES  IN  MAN  AND  ANIMALS  — 


I.   ACTINOMYCOSIS. 
II.  ACUTE  YELLOW  ATROPHY. 

III.  ANTHRAX. 

IV.  CATTLE  PLAGUE. 

V.   CEREBRO-SPINAL   MENINGITIS. 
VI.    CHICKEN-CHOLERA. 
VII.   CHOLERA. 
VIII.   DENTAL  CARIES. 
IX.    DIPHTHERIA. 
X.   ERYSIPELAS. 
XL    ENDOCARDITIS. 
XII.   GLANDERS. 

XIII.  GONORRHO2A. 

XIV.  HYDROPHOBIA. 
XV.    LEPROSY. 

XVI.  MALARIA. 
XVII.  MALIGNANT  CEDEMA. 
XVIII.   MEASLES. 


XIX.    OPHTHALMIC  DISEASES. 
XX.    OSTEO-MYELITIS. 
XXI.   PLEURO-PNEUMONIA. 
XXII.    PNEUMONIA. 
XXIII.    PUERPERAL   FEVER. 
XXIV.   PY^MIA  AND  SEPTICAEMIA. 
XXV.    RELAPSING  FEVER. 
XXVI.    RHINOSCLEROMA. 
XXVII.   SCARLATINA. 
XXVIII.    SWINE-ERYSIPELAS. 
XXIX.   SWINE-TYPHOID. 
XXX.   SYMPTOMATIC   ANTHRAX. 
XXXI.  SYPHILIS. 
XXXII.    TETANUS. 

XXXIII.  TUBERCULOSIS. 

XXXIV.  TYPHOID    FEVER. 
XXXV.   VARIOLA  AND  VACCINIA. 

XXXVI.   YELLOW   FEVER. 


K.    BACTERIA    IN    THE   AIR,    IN    SOIL,    AND    IN    WATER. 


(A)  METHODS. 

1852    PERTY. — Zur  Kenntniss  Kleinster  Lebensform. 
1867    PASTEUR. — Etudes  sur  la  Biere. 
1873    KLEBS. — Ueber    Fractionirte    Cultur.     Archiv      f. 
Exp.  Pathol,  Bd.  I. 

1875  BREFELD.— Verhandl.    d.    Physik.    Med.    Ges.    in 

Wiirzburg. 

COHN. — Beitrage  zur  Biologic  der  Pflanzen,  Bd.  I., 
Heft   3- 

1876  COHN. — Beitrage  zur  Biologic  der  Pflanzen,  Bd.  II., 

Heft  2. 

1877  SCHAFER. — Course  of  Practical  Histology. 
KOCH. — Verfahren  z.  Untersuch.,  z.  Conservir.  und 

Photographir.  der  Bakterien.     Beitrage  z.   Biol. 
d.  Pflanzen,  Bd.  II.,  Heft  3. 

1878  KOCH. — Untersuchungen      iiber      Wundinfections 

Krankheiten. 

1879  SALOMONSEN. — Zur    Isolation    Differenter    Bak- 

terien.    Bot.  Zeitung,  No.  39. 
BLANCHARD. —  Rev.  Inter.  Sci.,  III. 

1880  SALOMONSEN. — Eine  Einfache  Meth.  z.  Reincultur 

Versch.  Faulniss  Bakterien.     Bot.  Zeit.,  No.  28. 
EHRLICH. — Zeitschr.  f.  Klin.  Med.,  Bd.  I. 

1881  EHRLICH.— Zeitschr.  f.  Klin.  Med.,  Bd.  II.,  Heft  3. 
CARPENTER. — The  Microscope  and  its  Revelations. 

6th  Edition. 
KOCH. — Zur      Untersuchung     von       Pathogenen 

Organismen.     Mitth.    aus    d.    K.    Gesundheits 

Amt,  Bd.  I. 
BREFELD. — Bot.   Untersuch.    iiber  Schimmelpilze, 

Bd.  IV. 
DUCLAUX. — Ferments  et  Maladies. 

1882  KOCH. — Biol    Klin.  Woch.,  No.  15. 
EHRLICH. — Deut.  Med.  Woch.,  No.  19. 


372  APPENDIX. 

1882  FEHLEISEN. — Ueber  Neue  Method,  der  Untersuch. 

u.   Cultur  Pathogen.   Bakterien.     Physik.    Med. 
Ges.  zu  Wiirzburg. 

BuCHNER. — In   Nageli's  Untersuch.  iiber  Niedere 
Pilze.     Munich. 

1883  ALMQUIST. — Hygeia,  XLV.     Stockholm. 
BEHRENS. — Hilfsbuch  zur  Ausfiihrung  Mikroskop. 

Untersuch. 
HAZLEWOOD. — American      Monthly      Microscop. 

Journ. 
BREFELD. — Die      Klinstl.      Kultur     Parasitischer 

Pilze.  Botan.   Unters.  iiber  Hefenpilze,  Bd.  V. 
FLUGGE. — Handbuch     der      Hygiene     und     der 

Gewerbe  Krankheiten. 

1884  MAGNIN  and  STERNBERG. — Bacteria. 
FRIEDLANDER. — Microscopische      Technik.       ist 

Edition. 
BUCHNER. — Ueber    das   Verhalten   der    Spaltpilz- 

sporen  zu    den  Anilinfarben.     Aerztl.     Intelli- 

genzbl.,  No.  33. 

ORTH. — Patholog.  Anatom.  Diagnostik. 
PLAUT. — Farbungs     Methode    z.    Nachweis.     der 

Micro-organismen.      ist  Ed. 
BAUMGARTEN. — Beitr.     zur    Darstellungsmethode 

d.    Tuberkel  Bacillen.     Zeitschr.  f.  Wissensch. 

Mikr. 

KOCH. — Mitth.  a.  d.  Kais.  Ges.  Amt,  Bd.  II. 
GRAM. — Ueber  die  Isolirte  Farbung  der  Schizomy- 

ceten.     Fortsch.  d.  Med.,  II.,  No.  6. 

1885  HUEPPE. — Die     Methoden      der    Bakterien     For- 

schung. 

CORNIL  ET  BABES. — Les  Bacteries. 
GlBBES. — Practical  Histology  and  Pathology. 
HAUSER. — Ueber       Faulnissbacterien.       Mit     1 5 

Tafeln  in  Lichtdruck. 
KLEIN. —  Micro-organisms  and  Disease. 
LEE. — The  Microtomist's  Vade  Mecum. 


APPENDIX.  373 

1885  MALLEY. — Photomicrography. 

PLAUT. — Farbungs  Methoden.      2nd  Edition. 
WOODHEAD  and   HARE. — Pathological  Mycology. 
BizzoZERO       ET      FlRKET. — Manuel     de     Micro- 
scopic Clinique. 

JOHNE. — Ueber  die  Kochschen  Reinculturen. 
BORDONI  UFFREDUZZI. — Microparasitici. 
BANTI. — Manuale  di  Tecnica  Batteriologica. 
DOLLEY. — Technology  of  Bacteria  Investigation. 

1886  HUEPPE. — Die     Methoden     der     Bakterien     For- 

schung.      3rd  Ed. 

HUEPPE. — The  Methods  of  Bacteriological  Investi- 
gation. Translated  by  Biggs. 

HUBER  AND  BREKER. — Die  Path.  Histolog.  und 
Bacteriologischen  Untersuch.  Methoden. 

KLEIN. — Micro-organisms  and  Disease.      3rd  Ed. 

CORNIL  AND  BABES. — Les  Bacteries.     2nd  Edition. 

CROOKSHANK. — Manuel  Pratique  de  Bacteriologie, 
traduit  par  Bergeaud.  Avec  4  Photomicro- 
graphies. 

PLAUT. — Fortschr.  d.  Med. 

ElSENBERG. —  Bakteriologische  Diagnostik. 

ESMARCH. — Zeitschrift  f.  Hygiene. 

HUEPPE. — Bakteriologische  Apparate.  Deut.  Med. 
Woch. 


(B)  MORPHOLOGY  AND  CLASSIFICATION. 

1838    EHRENBERG. — Die  Infusionsthierchen  als  Volkom. 

Organism. 

1841     DujARDiN. — Histoire  Naturelle  des  Zoophytes. 
1866    DE    BARY. — Morph.   und    Phys.   der   Pilze. 
HALLIER. — Die  Pflanzlichen  Parasiten. 

1872  COHN. — Beitrage  zur  Biologic  der  Pflanzen,    Bd.  I. 

1873  LISTER. — Quart.  Journ.  Microscop.  Science. 
LANKESTER. — Quart.  Journ.  Microscop.  Science. 


374  APPENDIX. 

1874  BiLLROTH. — Unters.     liber     d.     Veg.    Form     der 

Coccobacteria    septica. 

1875  CORN. — Beitrage  z.  Biol.  d.  Pflanzen. 
DALLINGER  AND  DRYSDALE.    Monthly  Miscroscop. 

Journ. 
1877    LEUNIS. — Synopsis  d.  Pflanzenkunde.      Hanover. 

NAGELI. — Die  Niederen  Pilze. 
1879    VAN  TIEGHEM. — Compt.  Rend. 

1881  BREFELD. —  Botanische  Untersuch.  iiber  Schimmel- 

pilze,  Heft  I. 

KOCH. — Mitheil  aus  d.  K.  Ges.  Amt,   Bd.  I. 
RABENHORST. — Kryptogamenflora.     Bd.   I.,  Pilze, 

von  Winter. 

1882  SACHS. — Text-Book  of  Botany. 

1883  FLUGGE. — Handbuch  der  Hygiene. 

NEELSEN. — Neuere  Ansichten  iiber  d.  Systematik 

der  Spaltpilze.      Biol.  Centralbl.,  III.,  No.  18. 
VAN  TlEGHEM. — Traite  de  Botanique. 

1884  DE  BARY. — Verg.   Morph.   und   Biolog.  der  Pilze, 

Mycetozoen,  und  Bacterien. 
GROVE. — Synopsis    of    the     Bacteria    and    Yeast 

Fungi. 

MARPMANN. — Die  Spaltpilze. 
MILLER. — Deut.  Med.  Woch. 

1885  CORNIL  AND  BABES. — Les  Bacteries. 

FISCH. — Die    Systemat.    Stellung    der    Bacterien. 

Biolog.  Centralbl.,  V. 
ZOPF. — Die  Spaltpilze. 
A.  DE  BARY. — Vorlesungen  iiber  Bacterien. 
BIEDERT. — Virch.  Archiv,     Bd.  100. 
HAUSER. — Ueber  Faulniss  Bacterien. 

1886  HUEPPE. — Die  Formen  der  Bakterien. 
BAUMGARTEN. — Lehrbuch      der      Pathologischen 

Mykologie. 

FLUGGE. — Die  Micro-organismen.      2nd  Ed. 
LUTZ. — Fortschr.  d.  Med. 


APPENDIX.  375 

(C)   GENERAL  BIOLOGY. 
1877    NAGELI. — Die  Niederen  Pilze.     Munchen. 

1879  COHN  u.  MENDELSOHN. — Ueber  Einwirkung  des 

Elektrischen  Stromes   auf   die  Vermehrung  d. 
Bakterien.      Beitr.  z.  Biol.  d.  Pflanzen,  Bd.  III., 
Heft  I. 
NENCKI. — Virchow's  Archiv. 

1880  NENCKI. — Beitrage  zur  Biol.der  Spaltpilze.  Leipzig. 

1881  TUMAS. —  St.  Petersb.  Med.  Wochenschr. 
ENGELMANN. — Arch.  f.  d.  Ges.  Physiologic,  Bd.  26. 

1882  ENGELMANN. — Botan.  Zeitg. 

NAGELI. — Unters.  iiber  Niedere  Pilze.      Munchen. 

1884  REGNAKD. — Rech.   sur  1'Influence    de  tres-hautes 

Pressions  sur  les  Org.  Vivants.     Compt.  Rend., 

T.  98,  p.  744- 
CORTES. — De  1'Action  des  Hautes  Pressions  sur  les 

Phe'nomenes  de  la   Putrefraction,  etc.      Compt. 

Rend.  T.  99,  p.  385. 
NENCKI. — Ueber    das     Eiweiss    der     Milzbrand- 

bacillen.     Ber.  d.  Deutschen   Chem.  Gesellsch. 

S.  2605. 

1885  DUCLAUX. — Influence  de  la  Lumiere  sur  la  Vitalite 

des  Germes  de  Microbes.      Compt.  Rend. 
MlTTENZWEiG. — Die  Bakt.  Aetiolog.  der  Infect.  K. 

1886  V.  FODOR. — Bakterien    im   Blute  lebender  Thiere. 

Archiv.  f.  Hygiene. 
HAUSER. — Archiv.  f.  Exper.  Patholog.  u.  Pharma- 

cologie. 

WYSSOKOWITSCH. — Zeitschrift.  f.  Hygiene. 
PAVY. — Relation  of  Bacteria  to  Disease.     Harveian 

Oration.      Brit.  Med.  Journ. 
BORDONI-UFFREDUZZI. — Fortschr.  d.  Med. 
ARLOING. — Archiv.  de  Physiol. 
WATSON-CHEYNE. — Brit.  Med.  Journ. 
LlBORiUS. — Zeitschrift.  f.  Hygiene. 
DOWNES. — Action     of     Sunlight    on    Micro-org. 

Proc.  Roy.  Soc. 


376  APPENDIX. 

(D)  ZYMOGENIC  SAPROPHYTES  AND  FER- 
MENT A  TION. 

1860  MULLER. — Journ.  f.  Prakt.  Chem. 

PASTEUR. — Annal.  de  Chim.  et  de  Phys.,  III.  Ser., 

T.  58. 
PASTEUR. — Compt.  Rend.,  T.  50. 

1861  LlEBlG. — Verhandl.  der  Miinchener  Akad.  d.  Wiss. 
PASTEUR. — Bull,  de  la  Soc.  Chim. 

PASTEUR. — Compt.  Rend.,  T.  52. 

1862  PASTEUR. — Ann.  de  Chim.  et  de  Phys.,  T.  64. 
MONOYER. — These  de  Strassburg. 

1863  PASTEUR. — Compt.  Rend.,  T.  56. 

1864  PASTEUR. — Compt.  Rend. 

VAN  TiEGHEM. — Compt.  Rend.,  T.  58,  p.  210. 
TYNDALL. — Compt.  Rend.,  T.  58. 

1865  DUCLAUX. — Theses  presentees    a    la   Faculte    de 

Paris. 

BE"CHAMP. — Compt.  Rend.,  T.  60,  p.  445. 
TRE"CUL. — Ueber  Bacillus  Amylobacter.      Compt. 

Rend.,  T.  61. 

1866  PASTEUR. — Etudes  sur  le  Vin. 

1867  HALLIER. — Gahrungserscheinungen. 
TR£CUL.— Compt.  Rend.,  T.  65. 
TRECUL. — Ann.  des  Sc.,  Sen  7,  T.  7. 

1869  KARSTEN. — Chemismus  der  Pflanzenzelle. 
LlEBlG. — Verhandl.     der     Miinchener    Akad.     d. 

Wiss.,  5  Nov. 

1870  LlEBlG. — Ueber  Gahrung,  Quelle  der  Muskelkraft 

und  Ernahrung.      Leipzig  u.  Heidelberg. 

1871  PASTEUR. — Compt.  Rend. 
DUBRUNFAUT. — Compt.  Rend.,  T.  73. 
HoPPE-SEYLER. — Medic.-Chem.    Untersuchungen, 

Heft  4. 

1872  PASTEUR. — Compt.  Rend. 

DUMAS. — Compt.  Rend.,  T.  75,  Nr.  6. 
LEX. — Centralbl.  f.  d.  Med.  Wiss.,  S.  291. 


APPENDIX.  377 

1873  FlTZ.— Berichte  d.  Chem.  Ges.,  Bd.  6,  S.  48. 
HOFMANN. — Aerztl.  Verein  zu  Wien,  Mai. 
HOFMANN. — Allgem.  Med.  Centralbl.,  S.  605. 

1874  SCHUTZENBERGER. — Die  Gahrungserscheinungen. 
DUMAS. — Ann.  de  Chim.  et  de  Phys. 
BREFELD. — Landwirthsch.  Jahresber.,  Bd.  3. 
SCHEIBLER. — Ueber   die    Natur    des    Froschlaich. 

Zeitschr.  f.  Riibenzuckerindustrie. 
FELTZ  ET  RITTER. — Journ.  de  1'Anat.  et  Phys, 
HlLLER. — Centralbl.  f.  d.  Med.  Wiss.,  S.  53. 
VAN  TlEGHEM. — Compt.  Rend.,  T.  58. 
MUSCULUS. — Ber.  d.  Chem.  Ges.,  S.  124. 
MUSCULUS. — Compt.  Rend.,  T.  78. 

1875  POPOFF. — Botan.  Jahresber. 

BREP^ELD. — Landwirthsch.  Jahresber.,  Bd.  4. 
COLIN. — Bull,  de  TAcad.  de  Med. 

1876  PASTEUR. — Bull,  de  TAcad.  de  Med.,  No.  2  7. 
PASTEUR  ET  JOUBERT. — Compt.  Rend.,  T.  83. 
PASTEUR. — Etudes  sur  la  Biere. 

A.     MAYER. — Lehrbuch      der     Gahrungschemie. 

2  Aufl. 

FLECK. — Ber  d.  Chem.  Centralst.  Dresden. 
BREFELD. — Landwirthsch.  Jahresber.,  Bd.  5. 

1877  LISTER. — The   Cause  of  Putrefaction   and   Lactic 

Fermentation.     The  Pharmac.  Journ.  and  Tran- 
sact. 

HARZ. — Grundziige    der  alkoholischen  Gahrungs- 
lehre. 

1878  CIENKOWSKI. — Die    Gallertbildungen    d.    Zucker- 

riibensaftes. 
BOUTROUX. — Sur      la      Fermentation      Lactique. 

Compt.  Rend.,  T.  86. 
FlTZ. — Berichte  d.  Chem.  Ges.,  Bd.  TO,  p.  216. 

1879  FlTZ.— Berichte  d.   Chem.  Ges.,  Bd.    u,   pp.    42 

and  498  ;  and  Bd.  12,  p.  474. 
RlCHET. — Compt.  Rend.,  T.  88. 
NAGELI. — Theorie  der  Gahrung.     Miinchen. 


378  APPENDIX. 

1879  VAN      TiEGHEM. — Developpement    du     Spirillum 

Amyliferum.      Bull,  de  la  Soc.  Bot.  de  France, 

and     Sur    la    Fermentation    de     la    Cellulose, 

pp.   25—30. 

VAN  TIEGHEM. — Compt.  Rend.,  T.  88. 
VAN  TIEGHEM. — Identite  du  Bacillus  Amylobacter 

et  du  Vibrion  Butyrique  de  Pasteur.      Compt. 

Rend.,  T.  89. 

1880  FlTZ. — Berichte  d.  Chem.  Ges.,  Vol.  13,  p.  1309. 
PRAZMOWSKI.  —  Untersuchungen    iiber    die    Ent- 

wicklungsgeschichte        und       Fermentwirkung 
einiger  Bakterien. 
VAN  TIEGHEM. — Compt.  Rend. 

1881  B£CHAMP. — Compt.  Rend.,  T.  93. 

TYNDALL. — Essays  on  the  Floating  Matter  of  the 

Air. 
ERIKSSON. —  Unters.    aus    d.    Botan.     Institut    in 

Tubingen,  Heft  i. 
KERN. — Ueber   ein   neues   Milchferment  aus  dem 

Kaukasus.      Bull,  de  la  Soc.  Imp.  des  Natura- 

listes  de  Moskau,  No.  3. 
V.    JACKSCH. — Studien     liber    den  ,  Harnstoffpilz. 

Zeitschr.  f.  Physiol.  Chemie,  Bd.  5. 

1882  FITZ.— Berichte  d.  Chem.  Ges.,  Bd.  15,  p.  857. 

1883  GuiARD. — These  de  Paris. 

TAPPEINER.  —  Celluloseverdauung.     Fortschr.    d. 

Med.,  I.,  151  ;   II.,  377,  416. 
FlTZ. — Berichte  d.  Chem.  Ges.,  Bd.   1 6,  p.  844. 

1884  HUEPPE. — Unters.  iib.  die  Zersetzungen  der  Milch 

durch  Mikroorganismen.       Mitth.    a.     d.    Ges. 

Amt.,  Bd.  II. 

HUEPPE. — Deut.  Med.  Woch. 
KRANNHALS. — Ueber  das  Kumysahnliche  Getrank 

Kephir.      Deut.  Arch.  f.  Klin.  Med.,  Bd.  35. 
FlTZ. — Berichte  d.  Chem.  Ges.,   Bd.   17,  p.   1188. 
LADUREAU. — Sur  le Ferment Ammoniacal.    Compt. 

Rend.,  T.  99,  p.  877. 


APPENDIX.  379 

1884  VAN  TlEGHEM. — Developpement  de   1'Amylobac- 

ter  dans   les  Plantes  a  1'Etat  de  Vie  Normale. 
Compt.  Rend. 

1885  LUPINE  ET  Roux. — Compt.  Rend.,  T.  101. 
LEUBE. — Ueber   die    Ammoniakalische   Harngah- 

rung.     Virch.  Arch.,  Bd.  100,  S.  540. 
BILLET. — Sur  le  Bacterium  Ureae.   Ib.,  Bd.    100, 

p.  1252. 
SHERIDAN  LEA. — Journ.  of  Physiology. 


(E)   CHROMOGENIC    SAPROPHYTES. 

1833    HERMSTADT. — Ueber  die  Blaue  und  Rothe  Milch. 

1838  STEINHOFF. — Ueber   das   Blauwerden   der    Milch. 

Neue  Ann.  d.  Mecklenb.  Landw.  Ges. 

1839  EHRENBERG. — Micr.     Prodigiosus.     Verhandl.     d. 

Berl.  Acad. 
1850    CHABERT  ET  FROMAGE. — D'une  Alteration  du  Lait 

de  Vache,  designee  sous  le  Nom  du  Lait  Bleu. 
1852    GIELEN.— Mag.  f.  Ges.  d.  Thierheilkunde. 
i860    FORDOS. — Compt.  Rend,  de  1'Acad.  de  Sc. 

1862  LtJCKE.— Arch.  f.  Klin.  Chir. 

1863  EBERTH. — Centralbl.  f.  d.  Med.  Wissensch. 

1868  MOSLER. — Ueber  Blaue  Milch  und  durch-deren 
Genuss  herbeigefuhrte  Krankheiten.  Virchow's 
Archiv,  Bd.  43. 

1872  SCHROTER. — Ueber  einige  von  Bakterien  gebildete 

Pigmente.      Cohn,  Beitr.  z.  Biol.  der   Pflanzen, 
Bd.  I.,  Heft  2. 

1873  LANKESTER. — On     a    Peach-coloured    Bacterium. 

Quart.  Journ.  of  Micr.  Sc.,  Vol.  I  3. 
1875     FRANK. — Cohn's  Beitr.  z.  Biol.  d.  Pflanzen,  Bd.  I., 

Heft  3. 

GIRARD. — Unters.    iiber   Btauen    Eiter.      Chirurg. 
Centralbl.,  II. 


3  8o  APPENDIX. 

1875  KLEIN. — Quart.  Journ.  of  Micr.  Sc.,  Vol.  15. 

1876  LANKESTER. — Further  Observations  on   a   Peach- 

or    Red-coloured   Bacterium.     Quart.  Journ.    ol 
Micr.   Sc. 

1877  GIARD. — Revue  des  Sc.,  T.  5. 

1879  WERNICH. — Cohn's  Beitrage  zur  Biol.  d.  Pflanzen, 

Bd.  III.,  Heft  I. 

COHN   U.   MlFLET. — Cohn's  Beitrage   zur  Biol.   d. 
Pflanzen,  Bd.  III.,  Heft  i. 

1880  VAN  TIEGHEM. — Bull,  de  la  Soc.  Bot.  de  France. 
NEELSEN. — Cohn's  Beitr.  z.  Biologic  d.  Pflanzen, 

Bd.  III.,  Heft  2. 
l882    BABES. — Vom    Rothen     Schweiss.      Biolog.   Cen- 

tralbl.,  Bd.  2. 

GESSARD. — De  la  Pyocyanine  et  de  son  Microbe. 
1884    CHARRIN. — Communication     faite     a    la     Societe 

Anatomique. 
HUGUES. — Le  Lait  Bleu.      Echo  Veterinaire. 


(F)  SIMPLE  SAPROPHYTES. 

1833    KUTZING. — Sphaerotilus  Natans.    Linnaea,  8. 
1840    OERSTED.— Naturhist.  Tidsskrift,  Bd.  III. 
1842    GOODSIR. — Edinb.  Med.  and  Surg.  Journ. 
1845    WEISSE. — Monas   Okenii.      Bull.   Phys.-Mathemat. 

de  St.  Petersbourg,  III. 
1847    HELLER.— Heller's  Arch.  f.  Chemie. 

1858  EBERTH. — Virchow's  Archiv,  Bd.  I  3. 
ITZIGSOHN. — Virchow's  Archiv,  Bd.   13. 

1859  WELCKER. — Zeitschr.  f.  Ration.  Med. 

1861  MUNK. — Virch.  Arch.,  Bd.  22. 

1862  PASTEUR.— Ann.  de  Chim.  et  de  Phys.,  T.  64. 
1864    FRIEDREICH. — Beitrage  zur  Kenntniss  der  Sputa. 

Virch.  Arch.,  Bd.  30. 
MUNK. — Med.  Centralbl. 


APPENDIX.  381 

1865  COHN. — Ueber  zwei  Neue  Beggiatoen.     Hedwigia. 

1866  SURINGAR. — Arch.  Neerland. 

SURINGAR. — Ueber    den    Zellenbau   der    Sarcina. 
Bot.  Zeit. 

1867  LUDERS. — Ueber    Abstammung    u.    Entwicklung 

des  Bacterium  Termo. 

1871  LOSDORFER. — Med.  Jahrb.,  Heft  3. 

1872  COHN, — Beitr.  z.  Biol.  d.  Pflanzen,      Bd.  I. 

1873  LANKESTER. — Quart.  Journ.  of  Microscop.  Sc. 

1874  CASPARY. — Schriften   der  Physik.   Oekon.  Ges.   zu 

Konisberg,  Bd.  i  5. 
EWART. — Proceedings  of  the  Roy.  Soc. 

1875  ElDAM.   Cohn's  Beitr.  zur.  Biol.  d.  Pflanzen. 
DALLINGER  AND  DRYSDALE. — Monthly  Microsc. 

Journ. 

1876  LANKESTER. — Quarterly  Journ.  of  Microscop.  Sc. 

1877  HEIMER.  —  Ueber      Pneumonomycosis    Sarcinica. 

Deut.  Arch.  f.  Klin.   Med. 

1878  DALLINGER. — Journ.    of   the    Roy.    Micro.    Soc. 

London. 
GEDDES  AND  EWART. — On  the   Life-History  of 

Spirillum.   Proceed,  of  the  Roy.  Soc.  of  London. 
BREFELD. — Bacillus  Subtilis.      Ges.  Nat.  Freunde. 

1879  ZOPF. — Ueber  Crenothrix  Polyspora.      Berlin. 

1882  ZOPF. — Bacterium    Merismopedioi'des.       Sitz.-Ber. 

d.  Botan.  Ver.  d.  Prov.  Brandenb.     Juni. 
ENGELMANN. — Bacterium  Photometricum.   Unters. 

aus.   d.    Phys.   Laborat.   zu   Utrecht.      Pfliiger's 

Arch.  f.  d.  Ges.  Physiol,  Bd.  30,  S.  95. 
GlARD. — Sur    le    Crenothrix    Kiihniana.     Compt. 

Rend. 

1883  KURTH. — Bacterium  Zopfii.     Ber.  d.  Deutsch.  Bot. 

Ges.     Botanische  Zeitg. 

1884  VANDEVELDE. — Studien  zur  Chemie  des  Bacillus 

Subtilis.      Zeitschr.  f.    Phys.  Chemie,  Bd.  8. 
MULHAUSER.  —  Ueber    Spirillen.       Virch.    Arch., 
Bd.  97- 


382  APPENDIX. 

1885  FALKENHEIM. — Arch.  f.  Exp.  Patholog.  u.   Phar- 

makol.,  Bd.  19. 

BILLET. — Sur    la  Formation    des   Spores  chez  le 
Cladothrix  Dichotoma.      Compt.  Rend.,  T.  100. 

1886  BORDONI-UFFREDUZZI. — Ueber  d.  biol.    Eigensch. 

d.  norm.  Hautmikrophyt.    Fortsch.  d.  Med. 


(G)  PTOMAINES    AND    PUTREFACTION. 

1866  HEMMER.— Exper.  Studien  iiber  d.  Wirkung 
Faulender  Stoffe. 

SCHWENINGER. —  Ueber  d.  Wirkung  Faulender 
Org.  Substanzen. 

V.  RAISON. — Zur  Kenntniss  der  Putriden  In- 
toxication. 

BERGMANN. — Das  Putride  Gift. 

1868  BERGMANN  u.  SCHMIEDEBERG. — Med.  Centralbl. 

1869  ZULZER  u.  SONNENSCHEIN. — Berl.  Klin.  Woch. 

1872  BERGMANN. — Deut.  Zeitschr.  f.  Chirurgie,  Bd.  I. 
RAVITSCH. — Zur  Lehre  von  der  Putriden  Infection. 

1873  CLEMENTI  u.  THIN. — Unters.  lib.  d.  Putride  Infec- 

tion.    Wien.  Med.  Jahrb. 

1874  PANUM. — Virchow's  Arch.,  Bd.  60. 

FRISCH. — Exper.   Studien   ub.    d.    Verbreitung    d. 

Faulnissorganismen. 
KEHRER. — Archiv.  f.  Exper.  Pathol.,  Bd.  I. 

1876  HlLLER. — Centralbl.  f.  Chirurgie. 

NENCKI. — Ueber  die  Zersetzung  der  Gelatine  und 
des  Eiweisses  bei  der  Faulniss  mit  Pancreas. 

1877  SALOMONSEN. — Die  Faulniss  des  Blutes. 

1878  SELML— Chemische    Ber.,    Bd.    6,    7,     12,    Sulle 

Ptomaine  ad  Alcaloide  Cadaverici. 
KAUFMANN.  —  Zersetzung      des      Blutes      durch 
Bacillus     Subtilis.      Journ.    f.    Prakt.    Chemie, 
Bd.    17. 


APPENDIX.  383 

1879  HlLLER. — Die  Lehre  von  der  Faulniss. 

1880  HUSEMANN. — Arch.  d.  Pharmac. 

1881  KoNIG. — Massenerkrankung  von   Menschen   nach 

dem  Genuss  von  Fleisch  einer  an  Putrider 
Metritis  Verendeten  Kuh.  Ber.  lib  d.  Veteri- 
narwesen  im  Konigreich  Sachsen. 

TANRET. — Compt.  Rend.,  T.  92. 

BROUARDEL  ET  BOUTMY. — Compt.  Rend.,  T.  92, 
p.  1056. 

1882  HUSEMANN. — Arch.  d.  Pharmac. 

SCHIFFER. — Arch.    f.    Anat.   u.    Physiol.,   Physiol. 

Abtheil. 

BOCCI. — Centralbl.  f.  d.  Med.  Wiss. 
GROEBNER. — Beitrage  z.  Kenntniss  der  Ptomai'ne. 
BERGMANN  u.  ANGERER. — Das  Verhaltniss   der 

Fermentintoxication    zur     Septicamie.     Wu'rz- 

burger  Jubil.  Festschr. 
BOUCHARD. — Compt.  Rend,  de  Biol. 
NENCKI. — Journ.  f.  Pract.  Chem.,  Bd.  26. 
GAUTIER. — Compt.  Rend.,  T.  94. 
GAUTIER  ET  ETARD. — Compt.  Rend  ,  T.  94. 
ROSENBACH. — Giebt   es   Verschiedene   Arten    von 

Faulniss  ?      Deut.    Zeitschrift   fur    Chir.,  XVI., 

S.  342. 
ETARD  ET  OLIVIER. — De  la  Reduction  des    Sul- 

fates  par  les  Etres  Vivants.      Compt.  Rend. 

1883  HUSEMANN. — Arch  d.  Pharmac. 
GUARESCHI  ET  Mosso. — Arch.  Ital.  de  Biolog. 
BRIEGER. — Zur  Kenntniss    der    Faulnissalkaloide. 

Zeitschr.  f.  physiol.  Chemie,  Bd.  7. 

1884  MAAS. — Ueber     Faulnissalkaloide    des    gekochten 

Fleisches    und    des   Fischfleisches.    Fortschr.   d. 

Med.,  II.,  729. 

BRIEGER. — Ber.  d.   Deutsch.  Chem.  Ges.,  Bd.  17. 
VANDEVELDE. — Les  Ptomaines.     Arch,    de    Biol. 

par  van  Beneden. 
WILLGERODT. — Ueber  PtomaKne. 


384  APPENDIX. 

1884  BRIEGER. — Ueber  Giftige   Producte  der  Faulniss- 

bakterien.      Berl.  Klin.  Woch.,  Nr.  14. 
OTTO. — Anleitung  zur  Ausmittelung  der  Gifte. 

1885  TAPPEINER. — Med.  Centralbl. 

BLUMBERG. — Experimenteller  Beitrag  zur  Kennt- 
niss  der  Putriden  Intoxication.  Virch.  Arch., 
Bd.  100,  S.  377. 

OFFINGER. — Die  Ptomaine. 

BRIEGER. — Ueber  Ptomame. 

BRIEGER. —  WeitereUntersuchungen  iiberPtomai'ne. 

BACKLISCH. — Ber.  d.  Deutsch.  Chem.  Gesellsch., 
Bd.  1 8. 

HAUSER. — Ueber  Faulnissbakterien. 

1886  BRIEGER.— Ueber  Ptomaine.      Berl.  Klin.  Woch. 


(H)   ANTISEPTICS  AND  DISINFECTANTS. 

1875  ElDAM. — Einwirkung  Versch.  Temperaturen  a.  d. 

Entwicklung    von  Bacterium    Termo.       Cohn's 
Beitr.  zur.  Biol.,  Bd.  L,  Heft  3. 

SCHROTER. — Cohn's  Beitr.   zur  Biol.  der  Pflanzen, 
Bd.  L,  Heft  3. 

1876  KOCH.—  Cohn's  Beitr.   zur   Biol.  der   Pflanzen,  Bd. 

II.,  Heft  2. 

BUCHHOLZ. — Ueber  das  Verhalten  von   Bakterien 
zu  einigen  Antiseptics. 

1877  NAGELI. — Die  Niederen  Pilze. 
BUCHHOLZ. — Arch.  f.  Exp.  Pathol.,  Bd.  7. 
FRISCH. — Ueber  den  Einfluss   nied.   Temp.  auf.  d. 

Lebensfahigkeit  der  Bakterien.      Sitzungsber.  d. 

Wiener  Akad.,  Bd.  75  u.  80. 
TVNDALL. — Philos.  Transact,  of  the  Roy.  Soc. 
VALLIN.— Ann.  d'Hyg. 
1879    SOYKA. — Ber.  d.  Bayr.  Akad.  d.  Wissensch. 

SCHWARTZ. — Sitzungsber.  d.  Dorpater  Naturf.  Ges. 
WERNICKE. — Diss.  Dorpat. 


APPENDIX.  385 

1879  HABERKORN. — Das  Verhalten  von  Harnbakterien 

gegen  einige  Antiseptica.      Dissert.  Dorpat. 
WERNICH. — Die  Aromatischen  Faulnissproducte  in 
ihrer  Einwirkung    auf    Spalt  und    Sprosspilze. 
Virchow's  Archiv,  Bd.  78. 

1880  MERKE. — Virchow's  Archiv,  Bd.  81. 
MoRSCHELL. — Deut.  Med.  Woch. 
LASSAR. — Deut.  Med.  Woch. 
PASTEUR. — Ann.  d'Hyg. 

WERNICH. — Grundriss  der  Desinfectionslehre. 
MEYER. — Ueb.  d.   Milchsaureferment  u.  sein  Ver- 
halten gegen  Antiseptica. 
TOUSSAINT. — Bull,  de  1'Acad. 

1881  WOLFFHUGEL. — Ueber  den  Werth  der  schwefligen 

Saure  als  Desinfectionsmittel.      Mittheilg.   a.   d. 

Kais.  Ges.  Amt,  Bd.  I.,  S.  188. 
WOLFFHUGEL  u.  KNORRE. — Zu  der  verschiedenen 

Wirksamkeit    von     Carbolol    u.    Carbolwasser. 

Mitth.  a.  d.  Kaiserl.  Ges.  Amt,  Bd.  I.,  S.  352. 
KOCH. — Ueber   Desinfection.      Mitth.    a.  d.    Ges. 

Amt,  Bd.  I.,  S.  234. 
DE  LA  CROIX. — Das  Verhalten  der  Bakterien    d. 

Fleischwassers  gegen  einige  Antiseptica.     Arch. 

f.  Exp.  Pathol.,  Bd.  13. 
KOCH,  GAFFKY  u.  LOFFLER. — Versuche  iiber  die 

Verwerthbarkeit  heisser  Wasserdampfe  zu  Des- 

infectionszwecken.      Mitth.    a.  d.   Kaiserl.    Ges. 

Amt,  Bd.  I.,  S.  322. 

KOCH  u.  WOLFFHUGEL. — Unters.  iib.  die  Desin- 
fection mit  heisser  Luft.     Mitth.  a.  d.   Kaiserl. 

Ges.  Amt,  Bd.  I.,  S.  301. 
HUEPPE. — Ueber  das  Verhalten   ungeformter  Fer- 

mente  gegen  hohe  Temperatur.      Mittheilg.   a. 

d.  Kaiserl.  Ges.  Amt,  Bd.  I.,  S.  341. 

1882  LEBEDEFF. — Desinfectionsversuche    am    Malignen 

Oedem.     Arch,  de  Physiol.  Norm,  et  Pathol. 
W.  CHEYNE. — Antiseptic  Surgery. 

25 


386  APPENDIX. 

1882  LEBEDEFF. — Arch,  de  Phys.  Norm,  et  Pathol. 
HUEPPE. — Ueber  die  Hitze  als  Desinfectionsmittel. 

Deut.  Militararztl.  Zeitschr. 

1883  FISCHER. — Unters.  lib.  d.  Wirkung  des  Naphtalins. 

Berl.  Klin.  Woch. 
PERRONCITO. — Sur  la  Tenacite  de  Vie  du  Virus 

Charbonneux.     Arch.  Ital.  de  Biol. 
VALLIN. — Trait<£  des  Disinfectants  et  de  la  Disin- 
fection. 
MlQUEL. — Antiseptiques    et    Bacteries.       Semaine 

Medicale. 

CHAUVEAU. — Compt.  Rend. 
CHAMBERLAND  ET  Roux. — Compt.  Rend. 
LAILLIER. — Du    Gaz      Acide    Sulfureux.       Ann. 

d' Hygiene. 

ARLOING,  CORNEVIN,  ET  THOMAS. — Lyon  Med. 
VALLIN. — Les   Nouvelles  Etuves  a    Desinfection. 

Revue  d'Hygiene. 

LARRIVE". — L'Eau  Oxygenee.     These  de  Paris. 
MALY  u.  EMICH. — Antisept.  Wirkung  der  Gallen- 

sauren.     Sitzungsber.  d.  Kais.  Akad.  d.  Wiss.  zu 

Wien.  Jan. 
SCHULTZ. — Die  Antiseptischen  Eigenschaften  der 

Citronensaure.      Deut  Med.  Woch.,  Nr.  17. 

1884  SCHILL  U.  FISCHER. — Ueber  die  Desinfection  des 

Auswurfs  der  Phthisiker.      Mitth.  a.  d.   Kaiserl. 

Ges.  Amt,  Bd.  II. 

PLAUT. — Desinfection  der  Viehstalle. 
STEINMEYER. — Ueber  Desinfectionslehre. 
COLIN. — Compt.  Rend.,  T.  99. 
CHAIRY. — Action  des    Agents  Chimiques  sur  les 

BacteVies  du  Genre  Tyrothrix.      Compt.  Rend. 
CHAUVEAU. — Compt.  Rend. 
MlGNET. — Annuaire  de  TObservatoire   de    Mont- 

souris. 
SCHNETZLER. — Les    Proprie"te"s    Antiseptiques    de 

1'Acide  Formique.     Archiv.  de  Geneve. 


APPENDIX.  387 

1884  ROSSBACH. — Einfluss     des    innerl.    Naphthalinge- 

brauchs  auf  die  Harnfaulniss.     Berl.  Klin.  Woch. 
HOFFMANN. — Experimentelle         Untersuchungen 

iiber    die    Wirkung    der    Ameisensaure.     Diss. 

Greifswald. 
FISCHER   u.    PROSKAUER. — Mitth.    a.    d.   Kaiserl. 

Ges.  Amt,  Bd.  II. 
HEYDENREICH. — Sur  la  Sterilisation   des  Liquides 

au  moyen  de   la  Marmite   de  Papin.      Compt. 

Rend.,  T.  98. 
PICTET  ET  YOUNG. — De  1'Action  du  Froid  sur  les 

Microbes.      Compt.  Rend.,  T.  98. 
VALLIN. — Ann.  d'Hygiene. 
ROCHEFORT,  HERSCHER. — Revue  d'Hygiene. 
DUJARDIN-BEAUMETZ. — Bull.de  1' Acad.de  Med.  de 

Paris. 
MARIE-DAVY. — Revue  d'Hygiene. 

1885  M.  WOLFF. — Zur  Desinfectionsfrage.      Centralbl.  f. 

d.  Med.Wiss.,  Nr.  n. 
REINL. — Zur  Theorie  der  Heilwirkung  des  Fran- 

zensbader  Moores.      Prager  Med.  Woch.,  Nr.  10 

u.  II. 
THOL. — Ueber   d.    Einfluss    nicht    aromat.   organ. 

Sauren  auf  Faulniss  u.  Gahrung.      Diss.  Greifs- 
wald. 
SCHULZ. — Die    Ameisensaure     als    Antisepticum. 

Deut.  Med.  Woch.,  Nr.  24. 
SCHEDE. — Die  Antiseptische  Wundbehandlung  mit 

Sublimat.     Sammlung  Klin.  Vortrage,  Nr.  25. 
K6NIG. — Sublimatdampfe.     Chirurg.  Centralbl. 
FRANK. — Ueber  Desinfection  von  Abtrittsgruben. 
GARTNER  u.  PLAGGE. — Deut.  Med.  Woch. 

1886  HANDFORD. — Brit.  Med.  Journ. 

KOCH  u.  GAFFKY. — Arbeit,  a.  d.  K.  Gesundh.  Amt. 
WINTER  BLYTH. — Studies  of  Disinfectants  by  New 
Methods.     Proc.  Roy.  Soc. 


388  APPENDIX. 


(I)  IMMUNITY. 

1876    OEMLER. — Arch.  f.  Wiss.  u.  Pract  Thierheilk. 

1879  OLLIVE. — Sur   la  Resistance   des  Moutons  de  la 

Race    Berberine    a   1'Inoculation    du    Charbon. 
Compt.  Rend.,  T.  89. 

,         CHAUVEAU. — De  la  Predisposition  et  de  rimmu- 
nite  Pathologique.      Compt.  Rend.,  T.  89. 

1880  PASTEUR. — Bull,  de  1'Acad.  de  Med.  and  Gaz.  Med. 

de  Paris,  Nr.  18. 
SEMMER  u.    KRAJEWSKI. — Centralbl.   f.   d.    Med. 

Wiss. 
TOUSSAINT. — Bull,     de     1'Acad.     de    Med.  ;    and 

Compt.  Rend. 

1881  TOUSSAINT. — Compt.  Rend. 

LOEFFLER. — Zur  Immunitatsfrage.      Mitth.   a.   d. 

Ges.  Amt,  Bd.  I. 

SEMMER. — Virchow's  Arch.,  Bd.  83. 
GRAWITZ. — Die      Theorie      der      Schutzimpfung. 

Virchow's  Arch.,  Bd.  84. 

TOUSSAINT. — Gazette  Medicale  de  Paris,  Nr.  32. 
OEMLER. — Arch.  f.  Wiss.  u.  Pract.  Thierheilk. 

1882  R6SZAHEGYI. — Pester  Med.-Chir.  Presse. 
KOCH. — Ueber  die  Milzbrandimpfung. 

1883  FRANK. — Jahresber.     d.    K.     Thierarzneischule    in 

Miinchen. 

ARLOING,  CORNEVIN  ET  THOMAS. — Du  Charbon 
Bacterien;  Pathogenic  et  Inoculations  Pre- 
ventives. 

CHAUVEAU. — Compt  Rend.,  T.  96,  Nr.  9. 

CHAUVEAU. — Compt.  Rend.,  T.  96,  Nr.  10. 

CHAUVEAU. — Du  Role  de  1'Oxygene  de  1'Air  dans 
1'Attenuation  quasi-instantanee  des  Cultures 
Virulentes  par  1'Action  de  la  Chaleur.  Compt. 
Rend.,  T.  96,  Nr.  1 1. 


APPENDIX.  389 

1883  PASTEUR. — Sur     la     Vaccination    Charbonneuse. 

Compt.  Rend. 

PASTEUR. — La  Vaccination  Charbonneuse. 
PASTEUR. — Reponse    au    Doct.     Koch.        Revue 

Scientifique. 

PASTEUR. — Bull,  de  1'Acad.  de  Med. 
BUCHNER. — Eine  neue  Theorie  iiber  Erzielung  v. 

Immunitat  gegen  Infectionskrankheiten. 
PERRONCITO. — Sull'      Attenuazione     del     Virus 

Carbonchioso.      Atti  R.  Ace.  d.  Lincei. 
CHAMBERLAND  ET  Roux. — Compt.  Rend.,  T.  96, 

Nr.  15. 
CHAMBERLAND. — Le   Charbon   et   la  Vaccination 

Charbonneuse  d'apres  les  Travaux  Recents  de 

M.  Pasteur. 
MASSE. — Des    Inoculations    Preventives   dans    les 

Maladies  Virulentes. 

1884  FELTZ. — De   la    Duree  de    I'lmmunite"    Vaccinale 

Anticharbonneuse  chez  le  Lapin.  Compt. 
Rend.,  T.  99,  p.  246. 

CHAUVEAU. — De  1'Attenuation  des  Cultures  Viru- 
lentes par  I'Oxigene  Comprime'.  Gaz.  Heb- 
dom.  de  Med.  et  de  Chir.,  22. 

KOCH,  GAFFKY  u.  LOEFFLER. — Exper.  Studien 
iiber  d.  Kiinstl.  Abschwachung  der  Milzbrand- 
bacillen.  Mitth.  a.  d.  Ges.  Amt,  Bd.  II. 

BLAZEKOVIC — Zur  Praventiv-Inoculation  Pasteur's. 
Oesterr.  Monatschr.  f.  Thierheilk. 

PiiTZ. — Vortrage  f.  Thierarzte,  Ser.  7,  Heft  I. 

1885  HESS. — Vorl.    Mitth.    il   die  Schutzimpfung  gegen 

Milzbrand  im  Kanton  Bern  nach  der  Methode 

Chauveau.   Schweiz.  Arch.  f.  Thierheilk.,  Bd.  27. 
STREBEL. — Zur    Rauschbrandimpfung.       Schweiz. 

Arch.  f.  Thierheilk. 
BOULEY. — L'Inoculation   Preventive   de  la  Fievre 

Jaune.     Compt.  Rend.,  T.  100. 


3  go  APPENDIX. 

(J)  BACTERIA  ASSOCIATED    WITH  DISEASES 
IN  MAN  AND  ANIMALS. 

(I.)  Actinomycosis. 

1877  BOLLINGER. — Centralbl.  f.  d.  Med.  Wiss. 

1878  ISRAEL. — Virchow's  Arch.,  Bd.  74. 

1879  ISRAEL. — Virchow's  Arch.,  Bd.  78. 

1881  PONFICK. — Die  Actinomykose.      Berlin. 
JOHNE. — Deutsche  Zeitschr.  f.  Thiermed. 

1882  HiNK.— Centralbl.  f.  d.  Med.  Wiss. 
PFLUG. — Centralbl.  f.  d.  Med.  Wiss. 
GANNET. — Boston  Med.  and  Surg.  Journ. 

1883  PUSCH. — Arch.  f.  Wiss.  u.  Pr.  Thierheilk. 
BANG. — Tidskrift  far  Veterinaerer. 
ZEMANN. — Wien.  Med.  Jahrb.,  S.  477. 
FLEMING. — Actinomycosis. 

1884  ISRAEL. — Virchow's  Arch.,  Bd.  96. 
MITTELDORPF. — Deut.  Med.  Woch. 
KARSTEN. — Deut.  Med.  Woch. 
CHIARI. — Prager  Med.  Woch.,  Nr.  10. 
TREVES. — Lancet. 

FIRKET. — Rev.  de  Med. 

1885  ISRAEL. — Kenntniss       der       Actinomykose      des 

Menschen.      Klinische  Beitrage. 
BOSTROM. — Verh.  d.  Congr.  f.  Inn.  Med.  Wiesbaden. 
BAUMGARTEN. — Berl.  Klin.  Woch. 
JOHNE. — Bericht  u.  d.  Veter.-Wesen  i.  K.  Sachsen. 
MURPHY. — New  York  Med.  Journ. 
PONFICK. — Breslauer  Aerztl.  Zeitschr. 
SOLTMANN. — Breslauer  Aerztl.  Zeitschr. 
MAGNUSSEN. — Beitrage  zur  Diagnostik  u.    Casui- 

stik  der  Actinomykose.      Diss.   Kiel. 

1886  HERTWIG. — Archiv.  f.  Wiss.  u.  Prakt.  Thierheilk. 
O'NEILL. — Lancet. 

ACLAND. — Brit.  Med.  Journ.  and  Trans.  Path.  Soc. 
ROSER.— Deut.  Med.  Woch. 
ISRAEL. — Archiv  f.  Klin.  Chir. 


APPENDIX.  391 

(II.)  Acute   Yellow  Atrophy 

1875    EPPINGER. — Prag.  Viertelj. 
1882    HLAVA.— Prag.  Med.  Wochenschr. 

BALZER. — Archiv  de  Phys.  Norm.  et.  Path. 


(III.)  Anthrax. 

1855    POLLENDER. — Viertelj ahrschr.  f.   Ger.  Med.,  Bd.  8. 

1857  BRAUELL. — Virchow's  Arch.,  Bd.  n. 

1858  BRAUELL. — Virchow's  Arch.,  Bd.  14. 

1863    DAVATNE. — Compt  Rend.  Paris.     T.  57.     Ibid., 
T.  59- 

1865  DAVAINE. — Compt.  Rend.  Paris.  T.  60. 

1866  DAVAINE. — Compt.  Rend.  Paris.  T.  61. 

1872  BOLLINGER. — Centralbl.  f.  d.  Med.  Wiss.,  Bd.  10. 

1873  COLIN. — Bull.  Acad.  de  Med.,  Paris.  T.  2. 
DAVAINE. — Compt.  Rend.     Paris.  T.  77. 

1876  KOCH. — Beitrage  zur  Biologic  der  Pflanzen,  Bd.  II., 

Heft  2. 

1877  PASTEUR.— Compt.  Rend.     Paris.  T.  84. 
TOUSSAINT. — Compt.  Rend.     T.  85. 
DAVAINE. — Rec.  de  Med.  Vet.,  T.  4. 
DAVAINE. — Compt.  Rend. 

BERT. — Compt.  Rend.  Soc.  de  Biol.,  T.  4. 
PASTEUR. — Bull.  Acad.  de  Med. 

1878  COLIN. — Bull.  Acad.  de  Med.     Paris.  T.  7. 
TOUSSAINT — Compt.  Rend.     Paris. 

OEMLER. — Archiv   f.  Wiss.  u.    Pract.  Thierheilk., 

Bd.  4. 

EWART. — Quart.  Journ.  of  Microsc.  Sc. 
BERT.— Compt.  Rend.  Soc.  de  Biol.,  T.  5. 
KOCH. — Wundinfectionskrankheiten. 

1879  SCHMIDT. — Milzbrand  bei  Wildschweinen.     Deut 

Zeitschr.  f.  Thiermed.  u.  Vergl.  Pathol. 


3Q2  APPENDIX. 

1879  TOUSSAINT. — Recherches  Experimentales    sur    la 

Maladie  Charbonneuse. 
BERT. — Compt.  Rend.  Soc.  de  BioL,  T.  6. 
GREENFIELD. — Quart.  Journ.  Micr.  Sc.  London. 
OEMLER. — Archiv.  f.  Wiss.  u.  Pract.  Thierheilk. 
COLIN. — Bull.  Acad.  de  Med.      Paris.  T.  8. 
PASTEUR. — Bull.  Acad.  de  He'd.      Paris. 

1880  PASTEUR. — Compt.  Rend.      Paris.  T.  90  and  91. 
CHAUVEAU. — Compt.  Rend.      T.  90  and  91. 
TOUSSAINT.— Compt.  Rend. 

COLIN.— Bull.  Acad.  de  Med.      Paris.  T.  9. 
PASTEUR. — Bull.  Acad.  de  Med.      Paris. 
BOULEY. — Bull.  Acad.  de  Med.     Paris.  T.  9. 
WACHENHEIM. — Etude    Experimented     sur     la 

Septicite  et  la  Virulence  du  Sang  Charbonneux. 
BUCHNER.- — Ueber     die    Exper.     Erzeugung    des 

Milzbrandcontagiums  aus  den  Heupilzen. 
BUCHNER. — Versuche    iiber     die    Entstehung   des 

Milzbrands  durch  Einathmung.      Sitzungsber.  d. 

K.  Bayer.  Akad.  d.  Wissensch. 
FOKKER. — Centralbl.  f.  d.  Med.  Wissensch.  Bd.  I  8. 
SEMMER. — Centralbl.  f.  d.  Med.  Wiss..  Bd.  18. 
OEMLER. — Archiv  f.  Wissensch.  u.  Pract. Thierheilk. 
SZPILMAN. —  Zeitschr.  f.  Physiol.  Chemie.     Strass- 

burg.      Bd.  4. 
GREENFIELD. — Proc.  Roy.  Soc.  London. 

1881  PASTEUR. — Compt.  Rend.      Paris.   T.  92. 
CHAUVEAU. — Compt.  Rend.,  T.  92. 

BOULEY. — Compt.  Rend.     T.  92.     Ibid.,  T.  93. 
COLIN. — Bull.  Acad.  de  Med.,  T.  10. 
BOULEY.— Bull.  Acad.  de  Med.,  T.  10. 
RODET. — Contribution   a   1'Etude   Experimented 

du  Charbon  Bacteridien. 
KOCH. — Mitth.  aus.  d.  Ges.  Amt,  Bd.  I. 
FOKKER. — Centralbl.  f.  d.  Med.  Wiss. 
BUCHNER. — Vortrage  im  Aerztl.  Verein   zu   Miin- 

chen. 


APPENDIX .  3  93 

1881  HUBER.— Deut  Med.  Woch.,  Bd.  7. 

KLEIN. — Rep.  of  the  Medical  Officer  of  the  Local 

Govt.  Board. 
STERNBERG. — Am.  Monthly  Micr.  Journ. 

1882  CHAUVEAU. — Compt.  Rend.     Paris.  T.  94. 
RODET. — Compt.  Rend.,  T.  94. 
PASTEUR. — Compt.  Rend.      Paris.  T.  95. 
FELTZ. — Compt.  Rend.,  T.  95. 
FOKKER. — Virchow's  Archiv,  Bd.  88. 
ARCHANGELSKI. — Centralbl.  f.  d.  Med.  Wiss. 
SEMMER. — Der  Milzbrand  und  das  Milzbrandcon- 

tagium. 

ESSER  u.  SCHUTZ. — Zur  Casuistik  des  Milzbrands. 
Mitth.  a.  K.  Preuss.  Amtl.  Vet.  Sanitats- 
bericht. 

1883  CHAUVEAU. — Compt.  Rend.     Paris.   T.  96. 
ARCHANGELSKI. — Centralbl.  f.  d.  Med.  Wiss. 
ROLOFF. — Ueber    die    Milzbrandimpfung    und    d. 

Entwicklung  d.  Milzbrandbakterien.  Archiv 
f.  Wissensch  u.  Pract.  Thierheilk.,  Bd.  9. 

ROLOFF. — Der  Milzbrand.      Berlin. 

BiJCHNER. — Die  Umwandlung  der  Milzbrandbak- 
terien in  unschadliche  Bakterien.  Virchow's 
Arch.,  Bd.  91. 

TOEPPER. — Die  Neueren  Erfahrungen  iiber  d. 
Aetiologie  d.  Milzbrands. 

KLEIN. — Quart.  Journ.  Micr.  Sc. 

DOWDESWELL. — Rep.  Med.  Off.  Local  Gov.  Board. 

1884  BLEULER. — Milzbrand    beim     Menschen.       Corre- 

spondenzbl.  d.  Schweiz.  Aerzte. 
SCHRAKAMP. — Zur    Aetiologie     des    Milzbrandes. 

Archiv  f.  Hygiene,  Bd.  2. 
V.  CHELCHOWSKY. — Zur  Charakteristik  des   Milz- 

brandvirus.      Der  Thierarzt. 
SEMMER. — Centralbl.  f.  d.  Med.  Wiss.,  Bd.  22. 
PRAZMOWSKI. — Acad.  d.  Wissensch.  in  Krakau. 
PRAZMOWSKI. — Biol.  Centralbl.,  Bd.  4. 


394  APPENDIX. 

1885  BOLLINGER. — Zur     Aetiologie     des     Milzbrands. 

Sitzungsber.  d.  Ges.  f.  Morphol.  Physiol.  zu 
Miinchen. 

KlTT. — Sitzungsb.  d.  Ges.  f.  Morphol.  u.  Physiol. 
zu  Miinchen. 

FRIEDRICH. — Zur  Aetiologie  des  Milzbrands. 

OSOL. — Experiment.  Untersuch.  ii.  das  Anthrax- 
gift.  Inaug.  Diss.  Dorpat. 

1886  W.  KOCH. — Milzbrand  und   Rauschbrand.     Stutt- 

gart. 

HOFFA. — Die  Natur  des  Milzbrandgiftes.  Wies- 
baden. 

v.  FODOR. — Deut.  Med.  Woch. 

JOHNE. — Ber.  ii.  d.  Veter.Wesen.  i.  K.  Sachsen. 

BOLLINGER. — Arbeit,  a.  d.  Patholog.  Inst  zu 
Miinchen. 

FRANK. — Zeitschr.  f.  Hygiene. 


(IV.)  Cattle  Plague. 

1883  SEMMER  u.  ARCHANGELSKI. — Ueber  das  Rinder- 
pestcontagium  und  dessen  Mitigation.  Centralbl. 
f.  d.  Med.  Wiss. 


(V.)  Cerebro-Spinal  Meningitis. 
1883    LEYDEN. — Die    Mikrokokken    der    Cerebrospinal- 
Meningitis       Centralbl.  f.   Klin.  Med.,  Nr.  10. 
1885    LEICHTENSTERN. — Deut.  Med. Woch.,  Nr.  23  u.  3 1. 


(VI.)  Chicken-Cholera. 

1877  lOANNES    ET    MtfGNlN. — Journ.  d'  Acclimatation. 

1878  SEMMER. — Hiihnerpest.      Deut.  Zeitschr.  f.  Thier- 

med.  u.  Vergl.  Path. 


APPENDIX.  395 

1879  PERRONCITO. — Ueber  das  Epizootische  Typhoi'd 
der  Hiihner.  Arch.  f.  Wiss.  u.  Prakt.  Thierheilk. 

i860  PASTEUR. — Sur  le  Cholera  des  Poules.  Compt. 
Rend.,  T.  90. 

1882  ZiiRN. — Die  Krankheiten  des  Hausgefliigels. 
CoRNIL. — Arch,  de  Physiol.,  Bd.  10. 

1883  BARTH£L£MY. — De   1'Incubation   des  CEufs  d'une 

Poule  atteinte  du   Cholera  des  Poules.      Compt. 

Rend.,  T.  96.,  No,  18. 
BABES. — Arch,  de  Physiol. 
BABES. — Compt.  Rend,  de  1'Acad.  d.  Sc. 

1884  PETRL— Centralbl.  f.  d.  Med.  Wiss. 

1885  KlTT. — Mitth.     liber      die     Typhoi'dseuche      des 

Gefliigels.     Allg.  Deut.  Gefliigelzeitung. 


(VII.)  Cholera. 

1883  STRAUSS,  Roux,  THUILLIER  ET  NOCARD. — Compt. 

Rend.  Soc.  de  Biol.,  T.  4. 

1884  FlNKLER     U.     PRIOR. — Ueber     den    Bacillus    der 

Cholera   Nostras  und  seine  Cultur.      Naturfor- 

scherversammlung  Magdeburg. 
PFEIFFER. — Der  bisherige  Verlauf  der  Cholera  in 

Thiiringen,  etc.  Corresp.-Bl.  des  Allgem.  Aerztl. 

Ver.  in  Thiiringen,   Nr.  9. 
KOCH. — Ueber  die  Cholerabakterien.      Deut.  Med. 

Woch. 
EMMERICH. — Die  Cholera  in  Neapel.     Deut.  Med. 

Woch.,  Nr.  50. 
FINKLER  u.  PRIOR. — Unters.  iiber  Cholera  Nostras. 

Deut.  Med.  Woch.,  Nr.  36. 
DOYEN. — Mikro-organismen  in    Leber   und    Niere 

von   Choleraleichen.       Soc.   de   Biol.    de   Paris. 

Dec.  13. 
BUCHNER. — Ueber       Cholerabacillen.         Miinchn. 

Aerztl.  Intelligenzbl.,  S.  549. 


396  APPENDIX. 

1884  BIANCHI. — Lancet. 
MACNAMARA. — Brit.  Med.  Journ. 
HUNTER. — Brit.  Med.  Journal. 
CARTER. — Lancet. 
CAMERON. — Brit.  Med.  Journ. 
BOCHEFONTAINE. — Exper.    pour   servir  a   1'Etude 

des  Phenomenes  determines  chez  rHomme  par 
ringestion  Stomacale  du  Liquide  Diarrheique  du 
Cholera.     Compt.  Rend. 
LEWIS. — Med.  Times  and  Gazette.  September  2Oth. 

1885  JOHNE. — Einiges    iiber    die    sogen.    Choleracurse 

im.  K.  Ges.  Amt.      Deutsche  Zeitschr.  f.  Thier- 

med.,  Bd.  XI. 
FlNKLER  u.  PRIOR. — Forschungen  iiber  Cholera- 

bakterien.      Erganzungshefte  zum   Centralbl.  f 

Allg.      Gesundheitspflege,      Bd.     I.,      Heft      5 

u.    6. 

WATSON  CHEYNE. — Brit  Med.  Journ. 
BABES. — Virchow's  Archiv. 
FERRAN — Sur     1'Action     Pathogene    et    Prophy- 

lactique  du    Bacillus-virgule.      Compt.    Rend., 

T.  ioo,  p.  959. 
H^RICOURT. — Sur     la     Nature    Indifferente     des 

Bacilles-virgules.      Compt.  Rend.    ' 
VILLERS. — Sur  la  Formation  des  Ptomaines  dans 

le  Cholera.      Ibid.,  p.  91. 
FLUGGE. — Kritik    der    Emmerich'schen    Untersu- 

chungen    iiber    Cholera.       Deut    Med.   Woch., 

Nr.  2. 
PFEIFFER. — Ueber  die  Cholera  in   Paris.       Deut. 

Med.  Woch.,  Nr.  2. 
DENEKE. — Ueber  eine  Neue  den   Choleraspirillen 

ahnliche     Spaltpilzart.       Deut.     Med.     Woch., 

Nr.  3. 
MILLER. — Kommaformiger     Bacillus       aus       der 

Mundhohle.     Deut.  Med.  Woch.,  Nr.  9. 
KLEBS. — Ueber  Cholera  Asiatica. 


APPENDIX.  397 

1885  SCHOTTELIUS. — Zum  Mikrosk.  Nachw.  v.  Cholera- 

bacillen    in    Dejectionen.     Deut.    Med.   Woch., 

Nr.  14. 

DRASCHE. — Allg.  Wien.  Med.  Zeit. 
VAN   ERMENGEM. — Die  Ferran'schen    Impfungen. 

Deut.  Med.  Woch.,  Nr.  29. 
GIBIER  ET  VAN  ERMENGEM.     Rech.  Exper.  sur  le 

Cholera.     Compt.  Rend.,  T.  101. 
VAN  ERMENGEM. — Recherches  sur  le   Microbe  du 

Cholera  Asiatique. 

NlCATI  AND   RlETSCH. — Arch,  de  Physiol. 
NlCATl  AND  RlETSCH. — Revue  d'Hygiene. 
NlCATI  AND   RlETSCH. — Revue  de  Medecin,  T.  5. 
BRUNETTI. — Fatti   Considerazioni   Conclusioni  sul 

Colera. 
KLEIN. — Brit.    Med.   Journ.  and  Proc.  Roy.   Soc. 

London,  No.  38. 
KLEIN  and  GiBBES. — An  Inquiry  into  the  Etiology 

of  Asiatic  Cholera.      Bluebook. 
BUCHNER. — Archiv.  f.  Hygiene. 
BUCHNER  u.  EMMERICH. — Munch.  Med.  Woch. 
CROOKSHANK. — Remarks  on  the  Comma  Bacillus 

of  Koch.      Lancet. 

1886  KOCH.— Etiology    of    Cholera.       Berlin     Cholera 

Conference.  Translated  by  Laycock,  in 
Microparasites  and  Disease  (New  Syd.  Soc.) 

PETTENKOFER    (abstracted  by  Koplik). — Lancet. 

CANTANI. — Deut.  Med.  Woch. 

WEISSER. — Zeitschrift  f.  Hygiene. 

BITTER. — Archiv  f.  Hygiene. 

PFEIFFER. — Deut.  Med.  Woch. 

KLEIN. — Bacteria  of  Asiatic  Cholera.  Prac- 
titioner. 

WEISSER  and  FRANK. — Zeitschr.  f.  Hygiene. 


398  APPENDIX. 


(VIII.)  Dental  Caries. 

1867    LEBER    u.    ROTTENSTEIN. — Unters.   iiber   Caries 

der  Zahne.      Berlin. 
l880    ARNDT. — Beob.  an  Spirochaeta  Denticola.   Arch.  f. 

Pathol.  Anat,  Physiol.  u.  Klin.  Med.,  Bd.  79. 

1882  MILLER. — Der  Einfluss  der  Mikroorganismen   auf 

die  Caries  der  Zahne.    Arch.  f.  Exp.  Path.,  XVI. 

1883  MILLER. — Ueber    einen   Zahnspaltpilz   Leptothrix 

Gigantea.     Ber.  d.  Deutsch.  Bot.  Ges.,  Heft  5. 

1884  MILLER. — Deutsche  Med.  Woch.,  Nr.  25  u.  36. 
MILLER. — Correspdzbl.  f.  Zahnarzte,  Bd.  13. 
ROSENBACH. — Mikroorganismen  bei  den  Wundin- 

fectionskrankheiten,  S.  77. 


(IX.)  Diphtheria. 

1867  BUHL. — Micrococci  of   Diphtheria.      Zeitschr.  fur 

Biol. 

1868  HUETER    U.    TOMMASI. — Centralbl.     f.    d.     Med. 

Wissensch. 

1871  OERTEL.— Deut.  Arch.  f.  Klin.  Med.,  Bd.  8. 

1872  LETZERICH. — Virch.  Arch.,  Bd.  55. 
BlRCH-HlRSCHFELD. — Archiv  fur  Heilkunde. 

1873  EBERTH.  —  Der    Diphtheritische     Process.     Med. 

Centralbl.,  XI,  Nr.  8. 

1874  LETZERICH. — Mikrochemische    Erscheinungen  des 

Diphtheritispilzes.      Berl.  Klin.  Woch.,  XL 

1875  KLEBS.— Arch.  f.  Exp.  Pathol,  Bd.  4. 

1876  LETZERICH. — Virch.  Arch,  Bd.  68. 

1878  ZAHN. — Beitrage  zur  Pathol.  u.  Histol.  der   Diph- 

theric. 

1879  FRIEDBERGER. — Ueber  Croup  u.  Diphtheritis  beim 

Hausgefliigel.       Deut.  Zeitschr.  fur  Thiermed. 
u.  Vergl.  Pathol. 


APPENDIX.  399 

1879    NlCATl. — Compt.  Rend.,  T.  88. 

i860    NEUMAYER. — Neue  Thesen  zur  Diphtheritisfrage. 

1881  EVERETT. — Med.  and  Surg.  Reporter. 
TALAMON. — Bull,    de    la     Soc.     Anat.    de    Paris. 

T.  56. 

CORNIL. — Arch,  de  Physiol. 
OERTEL. —  Zur    Aetiologie     der    Infectionskrank- 

heiten. 

FORSTER. — Wien.  Med.  Woch. 
LUMNER. — Aerztl.  Int.  BL,  No.  31. 

1882  WOOD  AND  FORMAD. — Bull.  Nat  Board  of  Health, 

Wash,  and  Med.  Times  and  Gazette. 

SALISBURY. — Gaillard's  Med.  Journ.     New  York. 

GERHARDT  AND  KLEBS. — Verhandlung  des  Con- 
gresses fiir  Innere  Med. 

1883  FiiRBRINGER. — Virch.  Arch.,  Bd.  91. 
HEUBNER. — Die  Experimented  Diphtheric. 
GERHARDT  u.  KLEBS. — Verhandl.  d.  Congresses  f. 

Inn.  Med. 
FRANCOTTE. — La  Diphtheric. 

1884  EMMERICH. — Deut.  Med.  Woch. 

EMMERICH. — Compt.  Rendus  et  Memoires  du  V. 

Congres  Internat.  d'Hygiene. 
RIVOLTA. — Die   Diphtheric  der  Hiihner  im  Ver- 

gleich  zu  der  des  Menschen.      Giornale  de  Anat. 

Fisiol.  e  Patol.  delli  Anim. 

LOEFFLER. — Mittheil.  a.  d.  Kais.  Ges.  Amt,  Bd.  II. 
1886    LOEFFLER. —  Microparasites     and    Disease    [New 

Syd.  Soc.]. 


(X.)  Erysipelas. 

1868    HtfTER. — Med.  Centralbl.,  Nr.  34. 
1873    RAYNAUD. — Union  MeM 

ORTH. — Archiv.   f.  Exper.   Pathol.  u.   Pharmacol., 
Bd.  I. 


4OO  APPENDIX. 

1874  RECKLTNGHAUSEN  u.  LANKOWSKI. — Ueber  Ery- 

sipelas.    Virchow's  Arch.,  Bd.  60. 
LUKOMSKY. — Virchow's  Archiv,  Bd.  60. 

1875  TROSIER. — Bull.  Soc.  Anat.  de  Paris. 

BAADER. — Zur  Aetiologie  des  Erysipels.   Schweiz. 

Naturf.  Gesellsch. 
KLEBS. — Archiv  f.  Exper.  Pathol.  u.   Pharmacol., 

Bd.  4. 

1878  TlLLMANNS. — Verhandl.  d.  Deutsch.  Ges.  f.  Chi- 

rurgie. 

1879  TlLLMANS.  —  Experimentelle      u.      Anatomische 

Unters.  etc.      Archiv  f.  Klin.  Chirurgie,  Bd.  23. 

1880  WOLFF. — Virchow's  Arch.,  Bd.  81. 

1881  DUPEYRAT. — Recherches  Cliniques  et  Experimen- 

tales  sur  la  Pathogenic  de  1'Erysipele. 
FEHLEISEN. — Ueber    den    Erysipelaspilz.      Wiirz- 
burger  Phys.  Med.  Ges. 

1882  FEHLEISEN. — Deut.  Zeitschr.  f.  Chir.,  Bd.  16. 

1883  FEHLEISEN. — Die       Aetiologie       des      Erysipels. 

Berlin. 

1884  JANICKE  u.  NEISSER. — Exitus  Lethalis  nach  Ery- 

sipelas.     Centralbl.  f.  Chir.,  Nr.  25. 
RHEINER. — Beitr.  z.  Path.  Anat.  des   Erysipels  bei 
Gelegenheit  der  Typhusepid.  in  Zurich.      Vir- 
chow's Arch.,  Bd.  100,  Heft  2. 

1885  NEPVEN. — Des  Bacteries  dans  1'Erysipele. 
DENUCE. — Etude  sur  la  Pathogenic  et  1'Anatomie 

pathologique  de  1'Erysipele. 


(XL)  Endocarditis. 

1875  KOESTER. — Virchow's  Arch.,  Bd.  72. 

1876  BiRCH-HiRSCHFELD  u.  GERBER. — Archiv  d.  Heil- 

kunde. 

1877  WEDEL.— Berl.  Klin.  Wochenschr.. 


APPENDIX.  4O I 

1878    KLEBS. — Archiv  f.  Exper.  Pathol.,  Bd.  9. 

O.  ROSENBACH. — Ueber  Artificielle  Herzklappen- 
fehler.     Archiv  fur  Exper.  Pathol.,  Bd.  9. 

1880  HAMBURG. — Ueber  Acute    Endocarditis.     Berlin. 

Inaug.-Diss. 
GOODHART. — Trans.  Path.  Soc.,  Vol.  XXXI. 

1881  OBERBECK. — Casuistische  Beitrage  zur  Lehre  von 

der  Endocarditis  Ulcerosa.      Inaug.-Diss. 
LEYDEN. — Zeitschr.  f.  Klin.  Med. 
WEIGERT. — Virchow's  Arch.,  Bd.  84. 
KOCH.— Mittheil.  a.  d.  Kais.  Ges.  Amt,  Bd.  I, 
OSLER. — Trans.  Int.  Med.  Congress. 

1882  WlLKS. — Brit.  Med.  Journ. 

1883  KUNDRAT. — Sitz.-Ber.  d.  Kais.  Acad.  d.  Wissensch. 

zu  Wien. 

1884  BRAMWELL. — Diseases  of  the  Heart. 
BRISTOWE. — Brit.  Med.  Journ. 
GlBBES. — Brit.  Med.  Journ. 

1885  WYSSOKOWITSCH.  —  Centralbl.    f.    d,    Med.    Wis- 

sensch.,  Nr.  33. 

COUPLAND. — Brit  Med.  Journ. 
ORTH,    J. — Versammlung    Deutseher     Naturf.    zu 

Strasburg. 

NOCARD. — Recueil  de  Med.  Vet. 
OSLER. — Brit.  Med.  Journ. 
WEICHSELBAUM. — Wien.  Med.  Woch. 

1886  RlBBERT.— Fortsch.  d.  Med. 


(XII.)  Glanders. 

1882  LOFFLER  U.  SCHUTZ. — Ueber  den  Rotzpilz.    Deut. 

Med.  Woch.,  Nr.  52. 

BOUCHARD,   CAPITAN   ET    CHARRIN.  —  Bull,   de 
1'Acad.d.  Sc.,  Nr.  51. 

1883  ISRAEL. — Berl.  Klin.  Woch.,  Nr.  n. 
WASSILIEFF. — Deut.  Med.  Woch.,  Nr.  1 1. 

26 


402  APPENDIX. 

1883  MOLKENTIN. — Zur    Sicherstellung    der    Diagnose 

von  Rotz.     Inaug.-Diss. 
STRUCK. — Deut.  Med.  Woch.,  Nos.  51  u.  52. 
VULPIAN  AND  BOULEY. — Bull,  de  1'Acad.  de  MeU 
FROHNER.  —  Rotzige    Elephantiasis    des     Kopfes 

beim  Pferde.     Rep.  d.  Thierheilk. 

1884  KlTT. — Versuche  iiber  d.  Ziichtung  des  Rotzpilzes. 

Jahresber.  d.  Munchen.  Thierarzneisch. 
GRUNWALD. — Zur  DifFerentialdiagnose  des  Rotzes. 

Oesterr.   Monatsschr.  fur  Thierheilk.,  Nr.  4. 
WEICHSELBAUM. — Zur  Aetiologie  der  Rotzkrank- 

heit  des  Menschen.  Wiener  Med.  Woch., 2  I — 24. 
1886    LOEFFLER. — Arbeit,  a.  d.  K.  Gesundh.  Amt. 


(XIII.)  Gonorrhoea. 

1879  NEISSER. — Centralbl.  f.  d.  Med.  Wiss.,  Nr.  28. 
RETER. — Centralbl.  f.  d.  Med.  Wiss. 

1880  BUCKER. — Ueber  Polyarthritis  Gonorrhoica.  Diss. 
BOKAI. — Ueber  das  Contagium  der  Acuten  Blen- 

norrhce.     Allgem.  Med.  Centralzeitung,  Nr.  74. 
BOKAI  AND     FlNKELSTEIN. — Prager    Med.    Chir. 

Presse. 
WEISS. — Le  Microbe  du  Pus  Blennorrhagique. 

1881  AUFRECHT. — Pathologische  Mittheilungen. 
HAAB.  —  Der     Mikrokokkus     der     Blennorrhcea 

Neonator. 

HlRSCHBERG  U.  KRAUSE. — Zur  Pathologic  der 
Ansteckenden  Augenkrankheiten.  Centralbl. 
f.  Pract.  Augenheilk. 

1882  MARTIN. — Rech.  sur    les   Inflamm.   Me"tast.  a  la 

suite  de  la  Gonorrhee. 
KRAUSE.  —  Die     Mikrokokken     der     Blenorrhcea 

Neonator. 
LEISTIKOW. — Ueber  Bakterien  bei  den  Venerischen 

Krankheiten.    Charite'-Annalen,  7  Jahrg.,  S.  750 


APPENDIX.  403 

1882  BOCKHART. — Sitzungsbericht  d    Phys.    Med.    Ges. 

zu  Wiirzburg. 

1883  BOCKHART. — Beitrag     z.    Aetiologie   und    Pathol. 

des  Harnrohrentrippers.      Viertelj.  f.  Dermatol. 

und  Syph. 
ARNING. — Gonokokken  bei  Bartolinitis.     Viertelj. 

f.  Dermatol.  u.  Syph.,  S.  371. 
ESCHBAUM. — Beitr.    zur   Aetiologie  der  Gonorrh. 

Secrete.     Deut.  Med.  Woch.,  S.  187. 
NEWBKRRY. — Maryland  Med.  Journ. 
CAMPONA. — Italia  Medica. 
AUFRECHT. — Mikrokokken  i.  d.    Inneren  Organen 

bei  Nabelvenenentzundung  Neugeborener.  Cen- 

talbl.  f.  d.  Med.  Wiss.,  Nr.  16. 
PETRONE. — Sulla  Natura  dell'Artrite  Blenorragica. 

Rivista  Clin.,  No.  2. 

1884  CHAMERON. — Progre"s  Medical,  43. 
STERNBERG. — Med.  News,  Vol.  45,  Nr.  16. 
KAMMERER. — Ueber  Gonorrh.  Gelenkentzundung. 

Centralbl.  f.  Chirurgie,  Nr.  4. 

WELANDER. — Gaz.  Med.  de  Paris. 

KRONER. — Zur  Aetiol.  der  Ophthalmoblennorrhoea 
Neonator.  Naturforschervers.  in  Magdeburg, 
Arch.  f.  Gyn.,  XXV.,  S.  109. 

SANGER.  —  Ueber  Gonorrh.  Erkrankung  der 
Uterusadnexe.  Naturforschervers.  in  Magde- 
burg. Ibid.,  S.  126. 

OPPENHEIMER.  Naturforschervers.  in  Magdeburg. 
Ibid.,  S.  5  I. 

1885  FRANKEL. — Deut.  Med.  Woch.,  S.  22. 

BUMM. — Der  Mikroorganismus  der  Gonorrhoischen 

Schleimhauterkrankungen. 
LUNDSTROM. — Studier    ofver    Gonokokkus.    Diss. 

Helsingfors. 
E.  FRANKEL. — Mikrokokken  bei  Colpitis.     Deut. 

Med.  Woch.,  Nr.  2. 

1886  SMIRNOFF. — Vrach. 


404  APPENDIX. 


(XIV.)  Hydrophobia. 

1881  COLIN. — Bull.  Acad.  de  Med.  Paris,  T.   10. 
DOL^RIS. — Gaz.   Med.   de  Paris,  T.   3.       Tribune 

Med.  Paris,  T.  14. 
PASTEUR. — Compt.  Rend. 

1882  PASTEUR,  CHAMBERLAND,  Roux  ET  THUILLER. 

— Compt.  Rend. 
BERT. — Compt.  Rend. 

1883  GlBlER. — Compt.  Rend. 

1884  PASTEUR. — Nouvelle  Communication  sur  la  Rage. 

Ann.  de  Med.  Veterin. 

PERCHERON. — La    Rage  et    les    Experiences    de 
M.  Pasteur. 

PASTEUR. — Compt.  Rend. 

FOL. — Acad.  des  Sciences. 
1886    BABES. — In  Les  Bacteries. 

DOWDESWELL. — Journ.     Roy.     Micro.     Soc.     and 
Lancet. 

DOLAN. — Hydrophobia.       M.     Pasteur    and     his 
Methods. 

PASTEUR. — Compt.  Rend. 

VlGNAL. — Brit.  Med.  Journ. 

LANKESTER. — Nineteenth  Century,  No.  114. 

KERR. — Brit.  Med.  Journ. 

BAUER. — Munch.  Med.  Woch. 

FRISCH. — Wien.  Med.  Woch. 

HlME. — Experimental  Researches  concerning  Pas- 
teur's Prophylactic.     Lancet. 


(XV.)  Leprosy. 

1879    NEISSER. — Breslauer  Aerztl.  Zeitschr. 

NEISSER. — Jahresber.   d.   Schles.  Ges.  fur  Vaterl. 
Cultur. 


APPENDIX.  405 

1880  HANSEN. — Virchow's  Archiv. 
GAUCHER  ET  HILLAIRET. — Progres  Med. 

1881  NEISSER. — Virchow's  Archiv,  Bd.  84. 

CORNIL  ET  SUCHARD. — Ann.  de  Dermat.  et  Syph. 
VOSSIUS. — Uebertragungsversuche  von  Lepra  auf 

Kaninchen.     Ber.  iiber  d.  Ophthalmologencon- 

gress  in  Heidelberg. 

JOHN  HILLS. — On  Leprosy  in  British  Guiana. 
l8S2    KOBNER. — Uebertragungsversuche  von   Lepra  auf 

Thiere.     Virchow's  Arch. 
KOBNER. — Virchow's  Arch. 
HAUSEN,  ARMAUER. — Virchow's  Arch.,  Bd.  90. 

1883  DAMSCH. — Uebertragungsversuche  von   Lepra  auf 

Thiere.     Virch.  Arch.,  Bd.  92.      Centralbl.  f.  d. 

Med.  Wissensch.,  Bd.  2  I . 
KAPOSI. — Wiener  Med.  Woch. 
HILLIS. — Trans.  Path.  Soc. 
BABks. — Etude    Comparative   des  Bacteries  de  la 

Lepre  et  de  la  Tuberculose.     Compt.  Rend. 
MORETTI. — II  Primo  Caso  di  Lebbra  nelle  Marcho 

Confermato  dalla  Presenza  del  Bacillus  Leprae. 
MtiLLER. — Deut.  Archiv  f.  Klin.  Med.,  Bd.  34. 

1884  VlDAL. — La  L6pre  et  son  Traitement. 
ARNING. — Ueber   das   Vorkommen    des    Bacillus 

Leprae    bei    Lepra   Anaesthetica   s.   Nervorum. 
Virchow's  Archiv,  Bd.  97. 
THIN. — Brit.  Med.  Journal. 

1885  VlRCHOW. — Berl.  Klin.  Woch.,  Nr.  12. 

UNNA. — Ueber  Leprabacillen.     Deut.  Med.  Woch., 

Nr.  32. 
STEVEN. — Brit  Med.  Journ. 

1886  TOUTON. — Fortsch.  d.  Med. 
UNNA. — Deut.  Med.  Woch. 
NEISSER. — Virchow's  Archiv. 

UNNA  u.  LUTZ. — Dermatologische  Studien,  Heft  I. 
MELCHER  u.  ORTMANN. — Berl.  Klin.  Woch. 


406  APPENDIX. 


(XVI.)  Malaria. 

1879  KLEBS   u.   TOMMASI-CRUDELI.  —  Arch.    f.    Exp. 

Pathol.,  Bd.  2. 

1880  TOMMASI-CRUDELI. — Der    Bacillus     Malariae    im 

Erdboden  von  Selinunte  u.  Campobello.    Arch, 
f.  Exp.  Pathol.,  Bd.  12. 

CUBONI  AND  MARCHIAFAVA. — Neue  Studien  iib. 
d.  Natur  der  Malaria.  Arch.  f.  Exp.  Pathol., 
Bd.  13. 

1881  STERNBERG.  —  Bull.     Nat.    Board     of     Health. 

Washington. 
CUBONI    AND    MARCHIAFAVA.  —  Atti   della    R. 

Acad.  dei   Lincei. 
LAVERAN. — Compt.  Rend. 

1882  MARCHAND. — Zur  Aetiologie  der  Malaria.     Virch. 

Archiv,  Bd.  88. 

ZIEHL — Deutsch.  Med.  Woch.,  Nr.  48. 
ROSZAHEGYI. — Von    der    Ursache    des    Wechsel- 

fiebers.     Biol.  Centralbl.,  Bd.  2. 
TOMMASI-CRUDELI. — Die  Malaria  von  Rom. 
RICHARD. — Bestatigung   der   Laveranschen   Beob. 

Compt.  Rend.,  No.  8. 
LAVERAN. — Les  Parasites  du  Sang  dans  1'Impalu- 

disme.     Compt.  Rend.,  No.  17. 
BARDELS. — Gaz.  des  Hopit. 

1883  TORELLI. — La  Malaria  in  Italia. 

MAUREL. — L'Etiologie  et  la  Nature  du  Paludisme. 
Ann.  d'Hygiene. 

1884  KOTELMANN. — Der    Bacillus   Malariae   im   Alter- 

thum.     Virchow's  Arch.,  Bd.  97. 
GERHARDT. — Zeitschr.  f.  Klin.  Med.,  Bd.  7. 
SEHLEN. — Fortschr.  d.  Med.,  Bd.  II. 
LEONI.— Gazetta  Medica  di  Roma. 
MARCHIAFAVA    AND    CELLI.  —  Atti     della      R. 

Academia   dei   Lincei. 


APPENDIX.  407 

1884  MARIOTTI    E    CIARROCCHI. — Lo      Sperimentale, 

T.  54. 

TOMMASI-CRUDELI. — La  Production  Naturelle  de 
la  Malaria.  Conference  faite  a  la  8.  Sess.  du 
Congrds  Intern.  Med.  a  Copenhague. 

LAVERAN. — Traite*  des  Fievres  Palustres. 

1885  MARCHIAFAVA  u.  CELLI. — Neue  Untersuchungen 

iiber  die  Malariainfection.     Fortschr.  d.  Med., 
Bd.   3. 

1886  CAMILLO  GOLGI. — Archivio  per  le  Scienze  Mediche. 


(XVII.)  Malignant   CEdema. 

1862    DAVAINE. — Bull,  de  1'Acad.  de  Med. 
1877    PASTEUR. — Vibrion.  Septique.     Bull,  de  1'Acad.  de 
Med. 

1881  KOCH. — Mitth.  aus  dem  Ges.  Amt,  I.,  S.  54. 
GAFFKY. — Mitth.  as.  d.  K.  Ges.  Amt. 
PASTEUR. — Vibrion.  Septique.     Bull,  de  1'Acad.  de 

MeU 

1882  BRIEGER   u.    EHRLICH. — Berl.  Klin.  Wochenschr., 

Nr.  44. 

LEBEDEFF. — Versuche  iiber  Desinfection  bei  Malig- 
nen  Oedembacillen.  Arch,  de  Phys.  Norm,  et 
Path. 

1884  LUSTIG. — Zur  Kenntniss  Bakteriamischer  Erkran- 

kungen  bei  Pferden  (Malignes  Oedem). 
Jahresber.  d.  K.  Thierarzneischule  zu  Han- 
nover. 

KlTT. — Unters.  iiber  Malignes  Oedem  und  Rausch- 
brand  bei  Hausthieren.  Jahresber.  der  K. 
Thierarzneischule  in  Miinchen. 

1885  HESSE,  W.  u.  R.— Ueber  Ziichtung  der  Bacillen 

des  Malignen  Oedems.      Deut.  Med.  Woch. 


408  APPENDIX. 


(XVIII.)  Measles. 

1882  KEATING. — Phil.  Med.  Times. 

1883  CORNIL  ET  BABES. — Archiv  de  Phys. 


(XIX.)  Ophthalmic  Diseases. 

1872    ROTH. — Retinitis     Septica.       Virchow's     Archiv, 

Bd.  55. 
1879    BALOGH. — Sphaerobakterien    in     der    entziindeten 

Hornhaut.     Med.  Centralbl.,  XIV. 

1882  KAHLER. — Ueber     Septische     Retinitis.       Prager 

Zeitschr.  f.  Prakt.  Heilk.,  Bd.  I. 
SATTLER. — Unters.  iiber  das  Trachom.     Ber.  lib. 

d.  Ophthalmologen  Congress  zu  Heidelberg. 
MICHEL. — Graefe's  Archiv  f.  Augenheilkunde. 

1883  SCHLEICH. — Verh.  des  Ophthalmologen  Congr.  zu 

Heidelberg. 
BOCK. — Ueber  die  Miliare  Tuberkulose  der  Uvea. 

Virchow's  Arch.,  Bd.  91. 
KUSCHBERT    U.    NEISSER. — Zur    Pathologic    und 

Aetiologie    der    Xerosis    Conjunctivas.      Bresl. 

Aerztl.  Zeitschr.,  Nr.  4. 
SATTLER. — Ueber  d.  Natur  der  Jequirity-Ophthal- 

mie.     Zehender's  Klin.  Monatsblatt ;  and  Wien. 

Med.  Woch.,  Nr.  1 7. 
SATTLER  ET  DE  WECKER. — L'Ophthalmie  J6qui- 

ritique. 
CORNIL  ET  BERLIOZ. — Sur  I'Empoisonnement  par 

le  J<fquirity.     Compt.  Rend,  de  1'Acad.  d.  Sc. 

1884  v-  REUSS. — Pilzconcretionen  i.  d.  Thranenrohrchen. 

Wien.  Med.  Presse. 

GOLDZIEHER. — Streptothrix  Fcersteri  im  unteren 
Thranenrohrchen.  Centralblatt  f.  Prakt.  Augen- 
heilk. 


APPENDIX.  409 

1884  KRONER. — Zur  Aetiologie  der  Ophthalmoblenno- 

rrhce.    Verb.  d.  Naturforscher-Vers.  Magdeburg. 
KUSCHBERT. — Deutsch.  Med.  Woch.,  Nr.  21. 
VOSSIUS. — Berl.  Klin.  Wochenschr.,  Nr.  1 7. 
KLEIN. — Centralbl  f.  d.  Med.  Wiss.,  Nr.  8. 
VENNEMANN  ET  BRUYLANTS. — Le  J^quirity  et  son 

Principe  Pathogene. 

NEISSER. — Fortschr.  d.  Med.,  Bd.  2,  S.  73. 
SALOMONSEN. — Fortschr.  d.  Med.,  Bd.  2,  S.  78. 

1885  WlDMARK. — Hygeia.      Stockholm. 
DEUTSCHMANN. — v.  Graefe's  Archiv,  Bd.  XXXI. 

1886  GlFFORD. — Archiv  f.  Augenheilkunde. 

v.  ZEHENDER. — Bowman  Lecture.     Lancet 


(XX.)  Osteomyelitis. 

l873  COLLMANN. — Bakterien  im  Organismus  eines  an 
einer  Verletzung  am  Oberschenkel  verstorbenen 
Madchens.  Gottingen. 

1875  EBERTH. — Virchow's  Arch.,  Bd.  65. 

1876  FRIEDMANN. — Fall      von      Primarer      Infectib'ser 

Osteomyelitis.      Berl.  Klin.  Woch. 
SCHULLER. — Zur  Kenntniss  der  Mikrokokken  bei 
Acuter   Infectioser   Osteomyelitis.       Mikrokok- 
kenherde     im      Gelenkknorpel.       Centralbl.     f. 
Chirurgie,  Nr.  12. 

1882  FEHLEISEN. — Phys.  Med.  Ges.  Wiirzburg. 

1883  BECKER. — Vorl.    Mitth.  iiber  den  die  Acute  Infec- 

tiose  Osteomyelitis  erzeugenden   Mikroorganis- 
mus.      Deut.  Med.  Woch.,  and  Berl.  Klin.  Woch. 

1884  PEYROUD. — Compt.  Rend. 

KRAUSE. — Ueber  einen  bei  der  Acuten  Infectiosen 
Osteomyelitis  vorkommenden  Mikrokokkus. 
Fortschr.  d.  Med.,  Bd.  2. 

ROSENBACH. — Vorl.  Mitth.  iiber  die  die  Acute 
Osteomyelitis  beim  Menschen  erzeugenden 
Mikroorganismen.  Centralbl.  f.  Chirurgie. 


4IO  APPENDIX. 

1884  RODET. — Compt.  Rendus,  T.  99. 

1885  GARRE. — Fortschr.  d.  Med. 


(XXI.)  Pleuropneumonia. 

1879  SUSSDORFF. — Ueber  d.  Lungenseuche  des  Rindes. 

Deutsche  Zeitschr.  f.  Thiermed.  u.  Vergl.  Pathol. 

1880  BRUYLANTS    ET    VERRIERS.  —  Bull,    de   1'Acad. 

Belg. 

1882  PASTEUR. — Note  sur  la  Peripneumonie  Contagieuse 

des  Betes  a  Cornes.      Recueil  de  Med.  Vet 

1883  CORNIL  ET   BABES. — Arch,  de  Physiol.  Norm,  et 

Path.,  T.  2. 

1884  POELS  U.  NOLEN. — Centralbl.  f.  d.  Med.  Wiss.,Nr.  9. 
MAYRWIESER. — Ueber    Infectiosen  Bronchialcroup 

bei  Rindern.     Woch.  f.  Thierheilk.  u.  Viehzucht, 
19. 

1886    POELS  U.  NOLEN. — Das  Contagium  der  Lungen- 
seuche.     Fortsch.  d.  Med. 

POELS. — Septische  Pleuro-pneumonia  der  Kalber. 
Fortsch.  d.  Med. 


(XXII.)  Pneumonia. 

1878    KuHN. — Die    Contagiose    Pneumonic.       Deutsch. 
Arch,  f.'  Klin.  Med. 

1880  BRUYLANT  ET  VERRIERS. — Bull,  de  1'Acad.  Beige. 

1881  KiJHN. — Die  Uebertragbarkeit  Endemischer  Pneu- 

monieformen      auf     Kaninchen.      Berl.      Klin. 
Wochenschr.,   Nr.    38. 

1882  FRiEDLANDER.^-Virchow's  Arch.,  Bd.  87. 

1883  SALVIOLI   u.  ZASLEIN. — Ueber   den   Micrococcus 

und  die  Pathogenese  der  Crouposen  Pneumonic. 
Centralbl.  f.  d.   Med.  Wissensch. 
MATRAY. — Wien.  Med.  Presse,  Nr.  23. 


APPENDIX.  411 

1883  ZlEHL. — Ueber  das  Vorkommen  der  Pneumonie- 

kokken  im  Pneumonischen  Sputum.   Centralbl. 

f.  d.  Med.  Wissensch. 

FRIEDLANDER. — Fortschr.  d.  Med.,  Bd.  I. 
TALAMON. — Progr.  Medic. 
GILES.— Brit.  Med.  J.,  Vol.  II. 
FRIEDLANDER  u.  FROBENIUS. — Berl.  Klin.  Woch. 
GRIFFINI  AND   CAMBRIA. — Centralbl.   f.  d.  Med. 

Wiss. 

1884  FRIEDLANDER. — Fortschr.  d.  Med.,  Bd.  II. 
ZiEHL. — Ueber  das   Vorkommen  der  Pneumonie- 

kokken  im  Pneumonischen  Sputum.     Centralbl. 

f.  d.  Med.  Wissensch. 
KLEIN. — Centralbl.  f.  d.  Med.  Wissensch. 
NAUWERCK. — Ueber  Morbus  Brightii  bei  Crouposer 

Pneumonic.       Beitr.    zur     Pathol.    Anat.     von 

Ziegler. 

JiJRGENSEN. — Berl.  Klin.  Wochenschr.,  Bd.  22. 
MENDELSSOHN. — Zeitschr.  f.  Klin.  Med.,  Bd.  7. 
PLATONOW. — Ueber  die  Diagnostische  Bedeutung 

d.  Pneumoniekokken.  Inaug.-Diss.  Wiirzburg. 
KORANYi  u.  BABES. — Pester  Med.  Chirurg.  Presse. 
A.  FRANKEL. — Verhandl.  d.  Congr.  f.  Innere  Med., 

Fortschr.  d.  Med.      Nov. 

GERMAIN-SF!E. — Compt.  Rend.  Acad.  de  Sc.  Paris. 
AFANASSIEW. — Compt.   Rend.  Soc.  de  Biol.  Paris. 

T.  5. 

SALVIOLI  ET  ZASLEIN. — Arch,  pour  les  Sc.  Med., 

T.  8. 
MAGUIRE. — Brit.  Med.  Journ.,  Vol.  II. 

1885  DRESCHFELD. — Ueber    Wanderpneumonie  u.  ihre 

Beziehung     zur      Epidemischen      Pneumonic. 

Fortschr.  d.  Med.,  Bd.  3,   389. 
ScHOU. — Untersuchungen    iiber   Vaguspneumonie 

Fortschr.  der  Medicin,   Bd.  3,    Nr.  15. 
RIBBERT. — Zur     Farbung    d,    Pneumoniekokken. 

Deut.    Med.    Wochenschr.,    Nr.    9. 


412  APPENDIX. 

1885  GERMAIN-SE*E. — Des      Maladies     Specifiques     du 

Poumon. 

DE  BLAST. — Rivista  Internaz.  di.  Med.  e.  Chir. 
PAWLOWSKY. — Berl.  Klin.  Woch. 
PLATANOW. — Mitth.  a.  d.  Wiirzburg.  Med.  Klinik. 
STERNBERG. — Amer.  Journ.  Med.  Sciences. 
PETERLEIN. — Bericht  ii.  d.  Vet-Wesen.  i.  K.  Sach- 

sen. 
FRIEDLANDER. — Beste   Farbung    zur    Darstellung 

der  Kapseln  der  Pneumoniekokken.    Fortschritt, 

Bd.  3,  92. 

1886  STERNBERG. — Micrococcus       Pasteuri.         Journ. 

Royal    Microscop.    Soc. 
NEUMANN. — Berl.  Klin.  Woch. 
PIPPING. — Fortsch.  d.  Med.      Nos.  10  and  14. 
FRANKEL. — Zeitschr.  f.  Klin.  Med.    Bd.  X.  and  XL 
FRANKEL. — Deut.  Med.  Woch. 
FOA  u.  BORDONI-UFFREDUZZI. — Deut.  Med.  Woch. 
WECHSELBAUM. — Wien.  Med.  Woch. 
MANFREDI. — Fortsch.  d.  Med. 
JACCOUD. — La  France  Mddicale. 
LANCEREAUX    ET    BESAN^ON. — Archiv.   Gen.  de 

MeU 
THOST. — Deut.  Med.  Woch. 


(XXIII.)  Puerperal    Fever. 

1865  MAYRHOFER. — Vibrionen  als  Krankheitsursache 
des  Puerperalfiebers.  Monatsschr.  f.  Geburtsk. 
u.  Frauenkrankheiten,  Bd.  25. 

1872  WALDEYER. — Ueber  Vorkommen    von    Bakterien 

bei   der   Diphtheritischen  Form  des  Puerperal- 
fiebers.    Arch.  f.  Gynakologie,  III. 

1873  HEIBERG. — Die      Puerperalen      und    Pyamischen 

Processe. 
ORTH. — Virchow's  Arch.,  Bd.  5.8. 


APPENDIX.  4  1 3 

1873  RECKLINGHAUSEN  u.  LANKOWSKI. — Ueber  Ery- 
sipelas. Virchow's  Arch.,  Bd.  60. 

1879  LAFFTER. — Gehirnerweichungsherde  durch  Mikro- 

kokkenembolieen  bei  Puerperaler  Pyamie. 
Bresl.  Aerztl.  Ztg. 

1880  DOLERIS. — La  Fievre  Puerp^rale  et  les  Organismes 

Infect. 
PASTEUR. — Bull,  de  1'Acad.  de  Med.,  T.  9. 

1881  KAREWSKI. — Exper.  Unters.  iiber  die  Einwirkung 

Puerperaler  Secrete  auf  den  Thierischen 
Organismus.  Zeitschr.  f.  Geburtsh.  u.  Gyna- 
kologie,  Bd.  7. 

1884  AUFRECHT. — Die  Exper.  Erzeugung  der  Endo- 
metritis  Diphtherica  Puerperalis.  Naturforsch. 
Versamml. 


(XXIV.)    Pyaemia,    Septicaemia,    and 
Suppuration. 

1866  RINDFLEISCH. — Lehrb.  der   Pathol.   Gewebelehre. 

i.  Aufl.,  S.  204. 

1867  WALDEYER. — Zur  Pathol.  Anatomic  der  Wund- 

infectionskrankheiten.     Virchow's  Arch.,  Bd.  40. 
LISTER.— Lancet. 

1870  AINSTIE. — Lancet. 

1871  TlEGEL. — Ueber     d.       Fiebererregenden      Eigen- 

schaften     des     Microsporon     Septicum.      Bern. 
Diss. 

WALDEYER. — Mikrokokkencolonien  in  den  Paren- 
chymatischen  Organen.  Vortrag  i.  d.  Med. 
Ges.  zu  Breslau.  4  Aug. 

1872  BURDON-SANDERSON. — Trans.  Path.  Soc. 

1873  BlRCH-HlRSCHFELD. — Untersuchungen    iiber   Py- 

amie. 

HEIBERG. — Die  Puerperalen  u.  Pyamischen  Pro- 
cesse. 


414  APPENDIX. 

1874  TiEGEL. — Virchow's  Archiv,  Bd.  60. 

1875  BARTHOLD. — Pyaemisch-Metast.  Dissert.  Berlin. 
BURDON-SANDERSON. — Brit.  Med.  Journ. 

1877  BALFOUR. — Edinb.  Med.  Journ. 
LISTER. — Med.  Times  and  Gazette. 

1878  DOWDESWELL. — Quart.  Journ.  Micr.   Sc.   London, 

Vol.  1 8. 

KOCH. — Wundinfectionskrankheiten.     Leipzig. 
BASTIAN. — Brit  Med.  Journ. 

1880  FERRET. — De  la  Septicemie.     Paris. 
BECK. — Rep.  Loc.  Govt.  Board. 
DRYSDALE. — Pyrexia. 

1881  KOCH. — Mittheil.  d.  Kais.  Ges.  Amts,  Bd.  I. 
OERTEL. — Zur    Aetiologie    der    Infectionskrank- 

heiten. 

OGSTON. — Brit.  Med.  Journ.,  Vol.  I. 
BE"CHAMP. — Compt.  Rend. 

BECHAMP. — Trans.  Internat.  Med.  Cong.  London. 
HORSLEY. — R.ep.  Med.  Officer  Loc.  Govt.  Board. 
LISTER. — Quart.  Journ.  Microscop.  Science. 

1882  OGSTON. — Journ.  of  Anat.  and  Phys.,  Vol.  17. 
BRAIDWOOD  AND  VACHER. — Brit.  Med.  Journ. 
STERNBERG. — Amer.  Journ.  Med.  Sc., — John  Hop- 
kins' Univ.  Stud.  Biol.  Lab. 

DOWDESWELL. — Proc.  Roy.  Soc.  London,  Vol.  34. 

BABES. — Compt.  Rend. 

DRESCHFELD. — Brit  Med.  Journ. 

SUTTON. — Trans.  Path.  Soc. 

ROSENBACH. — Mikroorganismen  bei  den  Wund- 
infectionskrankheiten des  Menschen.  Wies- 
baden. 

ARLOING. — Recherches  sur  les  Septice*mies. 

WATSON-CHEYNE.— Trans.  Path.  Soc,,  XXXV. 

STEVEN. — Glasgow  Med.  Journal. 

1885    BASSET. — Ueber    Mikroorganismen     der    Eitrigen 
Zellgewebsentzundung    des    Menschen.      Fort 
schritte  d.  Med.,  Nr.  2. 


APPENDIX.  415 

1885    GARRE". — Zur    Aetiologie    acut    Eitriger    Entziin- 

dungen.      Fortschritte  d.  Med.,  165. 
PASSET. — Fortschritte  d.  Medicin,  Bd.  3. 
KLEMPERER. — Zeitschr.  f.  Klin.  Med. 
HOFFA — Fortsch.  d.  Med. 


(XXV.)  Relapsing  Fever. 

1873  OBERMEIER. — Vorkommen  Feinster,  Eigene 
Bewegung  zeigender  Faden  im  Blute  von 
Recurrenskranken.  Med.  Centralbl.,  1 1.  Berl. 
Med.  Ges.  Berl.  Klin.  Wochenschr. 
ENGEL. — Ueber  die  Obermeier'schen  Recurrens- 
spirillen.  Berl.  Klin.  Woch. 

1875  LAPTSCHINSKY. — Centralbl.  f.  d.  Med.  Wissensch., 

Bd.    13. 

1876  WEIGERT. — Deut.  Med.  Woch. 
HEYDENREICH. — St.  Petersb.  Med.  Woch. 
MANASSEM. — S.  Petersb.  Med.  Woch.,  No.  1 8. 

1877  HEYDENREICH. — Der  Parasit  des   Ruckfalltyphus. 

1879  CARTER. — Lancet. 

ALBRECHT. — St.  Petersb.  Med.  Woch. 
CORN. — Deut.  Med.  Woch. 
KOCH. — Deut.  Med.  Woch. 

1880  GUTTMANN. — Virchow's  Arch. 
MUHLHAUSER. — Virchow's  Arch.,  Bd.  97. 
JAKSCH. — Wien.  Med.  Wochenschr.     Juli. 

1885    GUNTHER. — Fortschr.  d.  Med. 

(XXVI.)  Rhinoscleroma. 

1885  CORNIL. — B.  de  la  Soc.  Anatom.      I  5  Fev. 
CORNIL  ET  ALVAREZ. — Sur   les  Micro-organismes 

du  Rhinosclerome.     Acad.  de   Me"d.  et  Archiv. 
de  Phys.  Norm,  et  Path. 

1886  PALTAUF  u.  EISELSBERG. — Fortsch.  d.  Med. 
DAVIES. — Brit.  Med.  Journ. 


416  APPENDIX. 

(XXVII.)  Scarlatina. 

1872    COZE  ET  FELTZ. — Les  Maladies  Infectieuses. 

McKENDRlCK. — Brit.  Med.  Journal. 

1876    KLEIN. — Report    of  the    Medical    Officer    of   the 
Privy  Council. 

1882  HAHN.— Berl.  Klin.  Woch.,  No.  38. 

1883  ROTH. — Miinchener  Aerztl.  Intelligenzbl. 
PoilL-PlNCUS. — Centralbl.  f.    d.    Med.   Wiss.,  No. 

36. 
CROOKE. — Lancet, 

1884  HEUBNER     u.     BAHRDT. — Zur     Kenntniss     der 

Gelenkeiterungen    bei    Scharlach.      Berl.    Klin. 
Woch.,  Nr.  44. 

1885  CROOKE. — Fortsch.  d.  Med. 

1886  LAURE. — Lyon  Medical. 
KLEIN.— Nature,  XXXIV. 

(XXVIII.)  Swine-Erysipelas. 

1881  SALMON. — Report  Depart.  Agricul.  Washington. 
DETMERS. — Science. 

1882  PASTEUR. — Sur    le   Rouget    ou    Mai    Rouge    des 

Pores.     Compt.  Rend.,  T.  95. 

1883  CORNIL  ET  BABES. — Arch,  de  Physiol. 
PASTEUR   ET   THUILLIER. — Bull,    de   1'Acad.  de 

Med.  de  Paris.     Compt.  Rend.,  T.  97. 
EGGELING. — Ueber  den  Rothlauf   der   Schwetne. 
Fortschr.  d.  Med. 

1884  SALMON. — Rep.  Dept.  Agricult.  Washington. 

1885  L6FFLER. — Experimentelle  Untersuchungen   iiber 

Schweinerothlauf.     Arbeiten  aus  dem  Kaiserl. 

Gesundheits  Amt,  Bd.  I. 
SCHUTZ. — Ueber  den  Rothlauf  der  Schweine  und 

die  Impfung  desselben.      Ibid. 
LYDTIN    u.    SCHOTTELIUS. — Der    Rothlauf   der 

Schweine.     Wiesbaden. 


APPENDIX.  4  I  7 


(XXIX.)  Swine-Typhoid. 

1879    KLEIN. — Rep.   of  the    Med.    Offic.    of  the    Privy 

Council  [1877-1878]. 
1884    KLEIN. — Virchow's  Archiv. 


(XXX.)  Symptomatic  Anthrax. 

1878  BOLLINGER  U.  FESER. — Wochenschr.  f.  Thierheil- 
kunde. 

1880  ARLOING,    CORNEVING     ET    THOMAS. — Compt. 

Rend. 

1881  ARLOING,   CORNEVING    ET   THOMAS. — Bull,    de 

1'Acad.  de  He'd,  and  Revue  de  Med. 

1883  ARLOING,  CORNEVING  ET  THOMAS. — Du  Charbon 

Bacterien,  Charbon  Symptomatique,  etc. 

1884  KlTT. — Unters.  iiber  Malignes  Oedem  und  Rausch- 

brand    bei     Hausthieren.        Jahresber.    der    K. 
Thierarzneisch.  in  Miinchen. 

BABES. — Journ.  de  1'Anatomie. 

NEELSEN  u.  EHLERS. — Ueber  den  Rauschbrand. 
Ber.  d.  Naturforsch.  Ges.  zu  Rostock. 

EHLERS. — Unters.  iib.d.  Rauschbrandpilz.     Inaug. 
Diss.  Rostock. 

ARLOING,   CORNEVING  ET  THOMAS. — Revue  de 

Med. 

1886  ARLOING,  CORNEVING  ET  THOMAS. — Chabert's 
Disease.  Transl.  by  Dawson  Williams  in 
Microparasites  and  Disease.  (New  Syd.  Soc.) 

HESS.- — Bericht   iiber    die   entschadigten    Rausch- 
brand u.  Milzbrandfalle  im  Canton  Bern. 

STREBEL. — Schweiz.  Archiv  f.  Thierheilk. 


4  1  8  APPENDIX. 

(XXXI.)  Syphilis. 

1872    LOSTORFER. — Arch.  f.  Dermat.  u.  Syph. 
1879    KLEBS.— Arch.  f.  Exp.  Pathol.,  Bd.   10. 

1881  AUFRECHT. — Centralbl.     f.    d.    Med.    Wissensch., 

Bd.  19. 

1882  BlRCH-HiRSCHFELD. — Bakterien  in   Syphilitischen 

Neubildungen.  Centralbl.  f.  d.  Med.  Wissen- 
sch., Nrs.  33,  34. 

PESCHEL. — Centralbl.  f.  Augenheilk. 

MARTINEAU  ET  HAMONIC. — De  la  Bacteridie 
Syphilitique.  Compt.  Rend.,  p.  443. 

MoRlSON. — Maryland  Med.  Journ. 

1883  LETNIK. — Wien.  Med.  Wochenschr. 

MORISON. — Maryland      Med.     Journ.     Baltimore. 
MORISON. — Wiener  Med.  Wochenschr. 
HEYDEN. — Preservation     de     la     Syphilis,      etc. 
Traduit  par  Roberts. 

1884  PETRONE.—  Gaz.  Medica  Ital. 

TORNEY  ET  MARCUS. — Compt.  Rend.,  p.  472. 
LUSTGARTEN. — Wien.  Med.  Woch.,  Nr.  47. 
KoNiGER. — Deut.  Med.  Woch.,  S.  816. 

1885  LUSTGARTEN. — Die  Syphilisbacillen. 
DOUTRELEPONT  u.  ScHUTZ. — Deut.  Med.  Woch., 

Nr.  19. 

ALVAREZ  ET  TAVEL. — Bull,  de  1'Acad.  de  Med.  et 
Archiv.  de  Phys.  Norm,  et  Path. 

DE  GlACOMI. — Neue  Farbungsmethode  der  Syphi- 
lisbacillen. Correspondenzbl.  f.  Schweizer 
Aerzte,  Bd.  15. 

GOTTSTEIN. — Fortschr.   d.   Med.,   Bd.    3,    S.    543. 

KLEMPERER.— Deut.  Med.  Woch. 

1886  EVE  and  LlNGARD. — Brit.  Med.  Journ. 
BlENSTOCK. — Fortsch.  d.  Med. 

KASSOWITZ  u.  HOCHSINGER. — Wien.  Med.  Blatter. 
DISSE  u.  TAGUCHI. — Deut.  Med.  Woch. 


APPENDIX.  4  I Q 

(XXXII.)  Tetanus. 

1884    CARLE      E     RATTONE.  —  Studio      Sperimentale 
sull'    Etiologia    del  Tetano.      Giorn.    della   R. 
Acad.  di  Medicina  di  Torino. 
VOGEL. — Drei     Falle     von    Infectiosem   Tetanus. 

Deut.  Med.  Woch.,  Nr.  31. 
NICOLAIER. — Deut.  Med.  Woch.,  Nr.  52. 
1886     ROSENBACH. — Archiv.  f.  Klin.  Chir. 


(XXXIII.)  Tuberculosis. 

1868    VlLLEMiN. — Etude  sur  la  Tuberculose. 

KLEBS. — Virchow's  Arch.,  Bd.  44. 
1873    KLEBS. — Arch.    f.    Exp.    Pathol.     u.    Pharmakol., 

Bd   I. 
1877    COHNHEIM, — Uebertragbarkeit    der    Tuberculose. 

Berlin. 
1879    BAUMGARTEN. — Berl.  Klin.  Woch.,  Bd.  17. 

1881  TOUSSAINT. — Compt.  Rend.,  T.  93. 
BAUMGARTEN. — Centralbl.      f.     d.     Med.     Wiss. 

Berlin.     Bd.  19. 

1882  BAUMGARTEN. —  Centralbl.    f.    d.     Med.     Wiss. 

Berlin.      Bd.  20. 

BAUMGARTEN. — Deut.  Med.  Woch.,    Bd.  8. 
W7AHL. — Zur     Tuberculosefrage.         Deut.      Med, 

Woch,  Nr.  46. 
VAN  ERMENGEM. — Le  Microbe  de  la  Tuberculose. 

Ann.  de  la  Soc.  Beige  de  Microscopic. 
HlLLER.  Deut.  Med.  Woch.,  Bd.  8. 
BALOGH. — Wien.  Med.  Woch. 
KOCH. — Die    Aetiologie   der  Tuberculose.       Berl. 

Klin.  Woch. 
FORMAD. — The  Bacillus  Tuberculosis.    The  Philad. 

Medical  Times. 
GREEN. — Lancet. 


42O  APPENDIX. 

1882  CREIGHTON. — Trans.  Path.  Soc. 
RINDFLEISCH. — Phys.  Med.Ges.  zu  Wiirzburg,Nr.  8. 
EWART. — Lancet. 

EHRLICH. — Deut.  Med.  Woch. 

FRANTZEL  u.  PALMERS. — Berl.  Klin.  Woch. 

AUFRECHT. — Centralbl.  fur  d.  Med.  Wiss. 

1883  SCHUCHARDT     U.    KRAUSE. — Fortschr.     d.    Med. 
KLEBS. — Arch.     f.    Exp.    Pathol.     u.    Pharmakol., 

Bd.  17. 

EHRLICH. — Deut.  Med.  Woch.,  Bd.  9. 
FRANTZEL  u.  PALMERS. — Berl.  Klin.  Woch. 
DE  GlACOMI. — Fortschr.  d.  Med.,  Bd.  I,  S.  145. 
MEISELS. — Wien.  Med.  Woch. 
RAYMOND. — Arch.  Ge*n.  de  Med.,  T.  1 1. 
DEMME. — Jahresber.  d.  Jennerschen   Kinderspitals. 

Bern. 

LYDTIN. — Badische  Thierarztl.  Mittheil. 
RIBBERT. — Ueber  d.  Verbreitungsweise  der  Tuber- 

kelbacillen   bei  Huhnern.     Deut.  Med.  Woch., 

Nr.  28. 

WECHSELBAUM. — Wien.  Med.  Jahrb. 
JOHNE. — Ber.  iib.  d.    Veterinarwesen    im   Konigr. 

Sachsen. 
BOLLINGER. — Munch.   Aerztl.      Intelligenzbl.,   Nr. 

1 6. 

JOHNE. — Die  Geschichte  der  Tuberculose. 
PUTZ. — Ueber  die  Beziehungen    der    Tuberculose 

des  Menschen  zu  der  der  Thiere. 
CELLI  and  GUARNERI. — Arch,  pour  les  Sciences 

M^dic. 

WEIGERT. — Deut.  Med.  Woch.     Nr.  24.^. 
NAUWERCK. — Deut.  Med.  Woch. 
SMITH. — Bristol.  Med.  Chir.  Journ. 
WILLIAMS. — Lancet. 
VERAGUTH. — Arch.  f.  Exp.  Path.  u.  Pharmakol., 

Bd.  1 6. 
SCHOTTELIUS. — Virchow's  Archiv,  Bd,  91. 


APPENDIX.  421 

1883    BROUILLY. — Note    sur    la    Presence  des    Bacilles 

dans   les    Lesions   Chirurgicales   Tuberculeuses. 

Rev.  de  Chin,  T.  3. 

SCHLEGTENDAL. — Fortschr.  d.  Med.,  Bd.  I. 
KUSSNER. — Beitr.  z.  Impftuberculose.     Deut  Med. 

Woch.,  Nr.  36. 

AUFRECHT. — Centralbl.  f.  d.  Med.  Wiss.,  Bd.  21. 
BOLLINGER. — Centralbl.  f.  d.   Med.  Wiss.,  Bd.  21, 

S.  600. 

CHEYNE. — Brit.  Med.  Journ.,  Vol.  I. 
CHEYNE. — Practitioner.     London.     Vol.  XXX. 
BOCK. — Virchow's  Archiv,  Bd.  91. 
DETTWEILER. — Berl.  Klin.  Woch.,  Bd.  21. 
DAMSCH. — Deut.  Med.  Woch.,  Nr.  1 7. 
BABES. — Der  erste  Nachweis  des  Tuberkelbacillus 

im  Harn.      Centralbl.  f.  d.  Med.  Wissensch. 
BABES. — Compt.  Rendus. 
ROSENSTEIN. — Centralbl.  f.  d.  Med.  Wiss. 
LEVINSKY. — Deut.  Med.  Woch.,  Bd.  9. 
LlCHTHEiM. — Fortschr.  d.  Med.,  Bd.  I. 
WEST.— Lancet,  Vol.  I. 
WEST. — Trans.  Path.  Soc. 
ZlEHL.  —  Bedeutung     der     Tuberkelbacillen     fur 

Diagnose  und    Prognose.       Deut.    Med.  Woch., 

Nr.   5. 
DlEULAFOY    ET    KRISHABER. Arch,    de     Physiol. 

Norm,  et  Path.,  T.  I. 
MALASSEZ  ET  VIGNAL.  —  Ibid.,  T.    II.     Compt. 

Rend.,  T.  97. 
KOCH. — Kritische     Besprechung     der    gegen     die 

Bedeutung    der     Tuberkelbacillen      gerichteten 

Publicationen.      Deut.  Med.  Woch.,  Nr.  10. 
VIGNAL. — Compt.  Rend.  Soc.  de  Biol.,  T.  5. 
GlBBES. — Lancet. 
KUNDRAT. — Wien.  Med.  Presse. 
LANDOUZY  ET  MARTIN. — Rev.  de  Med.,  T.  3. 
CHIARI. — Wien.  Med.  Presse. 


422 


APPENDIX. 


1883  COCHET. — Compt.  Rend.  Soc.  de  Biol.,  Paris,  T.  5. 
PFEIFFER. — Berlin.  Klin.  Woch.,  Bd.  21. 
LEUBE.  —  Sitzungsber.    der    Phys.-Med.    Soc.    zu 

Erlangen. 

SPINA. — Studien  u'ber  Tuberculose.      Wien. 
CRAMER. — Sitzungsber.    d.    Phys.    Med.    Soc.    zu 

Erlangen. 

MARCHAND. — Deut.  Med.  Woch.,  Nr.  15. 
HERON. — Lancet 
GREEN. — Brit.  Med.  Journ. 
BAUMGARTEN. — Centralbl.  f.  d.  Med.  Wissensch. 

Berlin.     Bd.  21. 

BAUMGARTEN. — Zeitschr.  f.  Klin.  Med.,  Bd.  6. 
SOMARI     E   BRUGNATELLI.  —  Redii    R.     Instit. 

Lombardo. 

1884  MALASSEZ  ET  VIGNAL. — Sur  le  Microorg.  de  la 

Tuberculose  Zoogloei'que.    Compt.  Rend.,  T.  99, 

p.  200. 
DOUTRELEPONT. — Die  Aetiologie  des  Lupus  Vul- 

garis.  Vierteljahrschr.  f.  Dermatologie  u.  Syphilis. 
CORNIL  ET    LELOIR. --  Recherches,    etc.,   sur    la 

Nature  du   Lupus.     Arch,   de    Physiol.    Norm. 

et  Pathol. 
BABES  ET  CORNIL. — Note  sur  les  Bacilles  de  la 

Tuberculose.      Journ.  de  1'Anat.  et  de  la  Physiol. 

Norm,  et  Pathol. 
BOULEY. — La  Nature    Vivante    de  la  Contagion. 

Contagiosite  de  la  Tuberculose.      Paris. 
D£j£RlNE. — Rev.  de.  Med.  Paris.      T.  4. 
ARLOING. — Compt.  Rend. 
BlEDERT. — Virchow's  Archiv,  Bd.  98. 
LEYDEN.   -   -  Klinisches     iiber     Tuberkelbacillen. 

Zeitschr.  f.  Klin.   Med ,  VIII. 
MULLER. — Ueber  den  Befund  von  Tuberkelbacillen 

bei    Fungosen   Knochen    u.    Gelenkaffectionen. 

Centralbl.  f.  Chir.,  3. 
ALBRECHT. — Arch.  f.  Kinderheilk.,  Bd.  5. 


APPENDIX.  423 

1884  SCHILL  u.  FISCHER. —  Mitth.  a.  d.  Kaiserl.  Ges. 

Amt,  Bd.  II. 
LUSTIG. — Ueber  Tuberkelbacillen  im  Blut  bei   an 

Allg.    Acuter     Miliartub.     Erkrankten.     Wien. 

Med.  Woch.,  Nr.  48. 
VOLTOLINI.  —  Ueber    Tuberkelbacillen    im    Ohr. 

Deut.  Med.  Woch.,  Nr.  31. 
STRASSMANN. — Virchow's  Archiv,  Bd.  96. 
KOCH. — Mittheilungen  aus  dem  Kais.    Ges.  Amt, 

Bd.  II. 
BAUMGARTEN. — Centralbl.  f.  d.  Med.  Wissensch. 

Berlin.      Bd.  22. 

GAFFKY. — Verhalten  der  Tuberkelbacillen  im  Spu- 
tum.  Mitth.  a.  d.  Kaiserl.  Gesundheitsamt,  Bd.II. 
NEGRI. — Colorations  des  Spores  dans  les  Bacilles 

de  la  Tuberculose.     Journ.  de  Microsc.,  T.  8. 
SUTTON. — Trans.  Path.  Soc.  London,  Vol.  XXXV. 
ANDREW. — Lancet. 
WILLIAMS. — Journ.  Royal  Microscop.  Soc. 

1885  DOUTRELEPONT.  —  Fall    von    Meningitis    Tuber- 

culosa  nach   Lupus,  Tuberkelbacillen  im   Blut. 

Deut.  Med.  Woch.,  Nr.  7. 
KARG. — Centralbl.  f.  Chir. 
SPINA. — Casopis  Lekaru  Ceskych,  Nr.  4. 
OBRZUT. — Prof.    Spina's    Neue    Farbungsmethode 

der  Faulnissmikroorganismen    und  ihre  Bezie- 

hung    zu  den    Tuberkelbacillen.       Deut.   Med. 

Woch.,  Nr.  12. 

CREIGHTON. — Brit.  Med.  Journ. 
CREIGHTON. — Lancet. 
FuTTERER. — Ueber  das  Vorkommen  u.   die   Ver- 

theilung  der  Tuberkelbacillen  in  den  Organen. 

Virch.  Arch.,  Bd.  100,  Heft  2. 
JOHNE. — Ein     Zweifelh.    Fall    von     Congenitaler 

Tuberculose.      Fortschr.  d.  Med.,  Bd.  3,  198. 
BlEDERT. — Berl.  Klin.  Woch. 
RIBBERT. —  Deut.  Med.  Woch. 


424  APPENDIX. 

1885  HARRIS. — St.  Barthol.  Hosp.  Reports. 
STICKER. — Centralbl.  f.  Klin.  Med. 
WESENER. — Fiitterungstuberculose. 
TSCHERNING. — Inoculationstuberculose  beim  Men- 

schen.      Fortschr.  d.  Med.,  Bd.  3,  65. 

1886  MlDDELDORPF. — Fortsch.  d.  Med. 
MuLLER. — Centralbl.  fur  Chir. 
GOLDENBLUM. — Vrach.,  Nos.  I.  and  XL 
BLACK. — Lancet. 

BAUMGARTEN. — Zeitschrift.  f.  Klin.  Med. 

KIRSTEIN. — Deut.  Med.  Woch. 

MAX  BENDER. — Ueber  die  Beziehungen  des  Lupus 

Vulg.  zu  Tuberc.      Deut.  Med.  Woch. 
HARRIES  AND  CAMPBELL. — Lupus.     London. 


(XXXIV.)  Typhoid  Fever. 

1874    BlRCH-HlRSCHFELD. — Unters.   zur  Pathologic  des 
Typhus   Abdominalis.      Zeitschr.   f.    Epidemio- 
logie,  I. 
KLEIN.— Med.  Centralbl.,  XII. 

1876  LETZERICH. — Virchow's  Archiv,  Bd.  68. 

1877  FELTZ.— Compt.  Rend.,  T.  85. 

1878  LETZERICH. — Archiv  f.  Exper.  Pathol.,  Bd.  9. 
l88o    EPPINGER. — Beitr.   zur    Pathol.    Anatomic   aus   d. 

Patholog.  Institut  Prag. 

FISCHEL. — Beitr.  zur  Pathol.  Anat.  aus  d.  Pathol.- 
Anat  Inst.  Prag. 

1880  TIZZONI. — Studi.    di    Pat.    Sperim.    sulla   Gen.   d. 

Tifo.      Milano. 

KLEBS. — Arch.  f.  Exper.  Pathol.  u.  Pharmakol. 
EBERTH. — Arch.  f.  Pathol.  Anat,  Bd.  81. 

1881  LETZERICH. — Archiv  f.  Exper.  Pathol.,  Bd.  10. 
BRAUTLECHT. —  Pathogene    Bakterien   im    Trink- 

wasser     bei     Epidemieen.       Virchow's     Arch., 
Bd.  84. 


APPENDIX.  425 

1881  MEYER. — Unters.   iiber  den  Bacillus  des  Abdomi- 

naltyphus.      Inaug.  Diss. 

RAPPIN. — Contrib.  a  1'Etude  des  Bact.  de  la  Bouche, 
a  1'Etat  Normal  et  dans  la  Fievre  Typhoi'de. 

1882  COATS. — Eberth's    Typhoid    Bacillus.      Brit.    Med. 

Journ. 

WERNICH, — Zeitschr.  f.  Klin.  Med.,  Bd.  6. 
MARAGHANO. — Centralbl.  f.  d.  Med.  Wiss.,  Bd.  15. 
ALMQUIST. — Typhoidfeberus-Bakterie. 
CROOKE. — Brit.  Med.  Journ. 

1883  BOENS. — Acad.  Roy.  de  Med.  de  Belgique.      Bull., 

3  Sen,  T.  17. 

EBERTH. — Der  Typhus- Bacillus  und  die  Inte- 
stinelle  Infection.  Volkmann,  Klin.  Vortrage. 

LETZERICH. — Experimentelle  Untersuchungen  iiber 
die  Aetiologie  des  Typhus  Abdominalis.  Leip- 
zig. 

1884  GAFFKY. — Zur  Aetiologie   des   Addominaltyphus. 

Mitth.  a.  d.  Ges.  Amt,  Bd.  II. 

TAYCN. — Le  Microbe  de  la  Fievre  Typhoide  de 
1'Homme.  Compt.  Rend.,  T.  99,  p.  331. 

1885  PFEIFFER. — Ueber    den    Nachweis    der    Typhus- 

bacillen   im   Darminhalt    u.  Stuhlgang.      Deut. 
Med.  Woch.,  Nr.  29. 

1886  MICHAEL.  —  Typhusbacillen       im       Trinkwasser. 

Fortsch.  d.  Med. 
MEISELS. — Wien.  Med.  WocL 
SEITZ. — Bakteriolog.  Studien  z.  Typhusatiologie. 
LUCATELLO. — Bollet  d.  R.  Accad.  Med.  di  Geneva. 
FRAENKEL     u.    SIMMONDS. — Die    Aetiologische 

Bedeutung  des  Typhus-Bacillus. 
NEUHAUSS. — Berl.  Klin.  Woch. 
SlROTININ. — Zeitschr.  f.  Hygiene. 
BEUMER  AND  PEIPER. — Zeitschr.  f.  Hygiene. 


426  APPENDIX. 

(XXXV.)  Variola  and  Vaccinia. 

l868    CHAUVEAU. — Nature    du    Virus-Vaccin.      Compt. 
Rend. 

1871  WEIGERT. — Ueber  Bakterien  in  der  Pockenhaut. 

1872  COHN. — Virchow's  Archiv,  Bd.  55. 
ZiJLZER. — Berl.   Klin.  Wochenschr. 

1873  LUGINBUHL. — Der      Micrococcus      der      Variola. 

Verhdl.  d.  Phys.  Med.  Ges.  in  Wiirzburg. 

1874  WEIGERT. — Anat.  Beitr.  z.  Lehre  v.  d.  Pocken. 
PlSSIN. — Berl.  Klin.  Wochenschr. 

STROPP. — Vaccination  u.  Mikrokokken. 

KLEBS. — Arch.  f.  Exp.  Path.  u.  Pharmakol.,  Bd.  i  o. 

1880  ISCHAMER. — Ueber    das    Wesen    des    Contag.   der 

Variola  Vaccine    u.   Varicella.     Aerztl.  Verein. 
Steiermark. 

1881  TAPPE. — Aetiologie  u.  Histologie  der  Schafpocken. 

1882  POHL-PlNCUS. — Vaccination. 

PFEIFFER. — Ueber  die  Ruckimpfung  auf  Kiihe 
und  Kalber.  Jahrb.  f.  Kinderheilk. 

MARCHAND. — Les  Virus-Vaccins.  Revue  Myco- 
logique. 

STRAUSS. — Vaccinal  Micrococci.  Soc.  de  Biol.  de 
Paris. 

1883  WOLF. — Zur  Impffrage.    Berl.  Klin.  WTochenschr., 

Nr.  4. 

PLAUT. — Das  Organis.  Contagium  der  Schafpocken. 
CORNIL  ET  BABES. — Soc.  Medicale  des  Hopitaux. 
QuiST. — St.  Petersburg  Medic.  Woch.,  Nr.  46. 

1884  HAMERINK. — Ueber     die     sog.     Vaccination      u. 

Variola. 


(XXXVI.)  Yellow  Fever. 

1883  BABES. — Sur  les  Microbes  trouves  dans  le  Foie  et 
dans  le  Rein  d'Individus  morts  de  la  Fievre 
Jaune.  Compt.  Rend.,  17  Sept. 


APPENDIX.  427 

1884  DOMINGOS   FREIRE    ET   REBOURGEON. — Compt. 

Rend.,  T.  99,  p.  804. 

DOMINGOS  FREIRE. — Recherches  sur  la  Cause  de 
la  Fievre  Jaune. 

1885  BOULEY. — L'Inoculation    Preventive   de   la   Fievre 

Jaune.     Compt.  Rend.,  T.,  100,  p.  1276. 
CARMONA  Y  VALLE. — Lemons  sur  1'Etiol.  et  la  Pro- 
phylax.  de  la  Fievre  Jaune. 

1886  CERECEDO. — El  Siglo  Medico. 

(K.)  BACTERIA  IN  THE  AIR,  IN  SOIL,  AND  IN 

WA  TER. 

1858     POUCHET. — Compt.  Rend.,  T.  47. 

1860  PASTEUR. — Compt.  Rend.,  T.  50. 

1861  PASTEUR. — Compt.  Rend.,  T.  52. 

1862  PASTEUR. — Ann.  de  Chim.  et  de  Phys.,  T.  64. 

1863  PASTEUR. — Compt.  Rend.,  T.  56. 
LEMAIRE. — Compt.  Rend.,  T.  57. 

1870    TYNDALL.— Med.  Tim.  and  Gaz. 

MADDOX. — Month.  Microscop.  Journal. 
1874    CUNNINGHAM. — Micr.  Exam,  of  the  Air.    Calcutta. 

TISSANDIER. — Comp.  Rend.,  T.  78. 

1877  PASTEUR. — Compt.  Rend.,  T.  85. 

MIQUEL. — Annuaire  de  1'Observat.  de  Montsouris. 
TYNDALL. — Brit.  Med.  Journ. 

1878  MIQUEL. — Compt.  Rend.,  T.  86. 

MIQUEL. — De  la  Presence  dans  1'Air  du  Ferment  de 
1'Uree.      Bull,  de  la  Soc.  Chim. 

1879  MlFLET, — Unters.   iiber   die  in  der    Luft   Suspen- 

dirten    Bakterien.       Cohn's    Beitr.    z.   Biol.    d. 

Pflanzen,  Bd.  III. 
WERNICH. — Cohn's  Beitrage  z.   Biol.  d.  Pflanzen, 

Bd.  III.,  S.  105. 
KLEBS  u.  TOMMASI-CRUDELI. — Unters.  der  Luft 

auf  die  Mikroorganismen  der  Malaria.     Archiv 

f.  Exper.  Path.,  Bd.  IT. 


4  2  8  APPENDIX. 

1879    MlQUEL- — Ann.  d'Hygiene. 

1881  KOCH. — Mtlzbrandbacillen  im   Boden.  Mitth.  a.  d. 

Ges.  Amt,  Bd.  I. 
SOYKA. — Sitz.-Ber   der   K.   Bayr.  Akad.   d.    Wiss. 

Math.  Physik.  Classe. 

SOYKA. — Vortrage  im  Aerztl.  Verein  in  Miinchen. 
BUCHNER. — Die     Bedingungen     des     Uebergangs 

von     Pilzen   in   die   Luft.   Vortrage   im   Aerztl. 

Verein  zu  Miinchen. 
TYNDALL. — Essays  on  the  Floating  Matter  of  the 

Air. 

1882  WlNNACKER. — Ueber  die  in  Rinnsteinen  beobacht. 

Nied.    Organisrnen.          Gottinger     Inaug.-Diss. 

Frankfurt  a.  M. 
V.  FODOR. — Hygienische  Unters.  iiber  Luft,  Boden 

u.  Wasser. 

NAGELI. — Unters.  Ciber  Niedere  Pilze. 
SOYKA. — D.  Vierteljsch.  f.  OefT.  Ges.,  Bd.  14. 
MIQUEL. — Annuaire  de  TObservat.   de  Montsouris. 
.1883     WOLLNY. — Ueber  die  Thatigkeit  Niederer   Orga- 

nismen  im  Boden.      Viert.  f.   Oeff.  Ges.,  S.  705. 
LETZERICH. — Exp.  Unters.   iib.  die  Aetiologie  des 

Typhus  mit  bes.  Berucksichtigung  der  Trink-  u. 

Gebrauchswasser. 
ZANDER. — Centralbl.  f.  Allg.  Ges. 
GUNNING. — Beitr.  z.   Hygienischen   Untersuchung 

des  Wassers,  Arch.  f.  Hyg.,  3. 
MIQUEL. — Les   Organismes    Vivants    de   1'Atmo- 

sphere. 
OLIVIER. — Les    Germes    de     1'Air.    These.     Rev. 

Scientif. 

ANGUS  SMITH. — Sanitary  Record. 
SMART. — Germs,  Dust,  and  Disease. 
TORELLI. — La  Malaria  in  Italia. 
HESSE. — Ueber  Quantitative   Best,  der  in  der  Luft 

enthaltenen    Mikroorganismen.        Mitth.    a.    d. 

Ges.  Amt,  Bd,  II. 


APPENDIX.  429 

1884  HESSE. — Ueber  Abscheidung  d.   Mikroorganismen 

aus  d.  Luft.     Deutsche  Med.  Wochenschr.,  2. 
HESSE. — Weitere  Mitth.  iiber  Luftfiltration.    Deut. 

Med.  Woch,  Nr.  51. 

SCHRAKAMP. — Archiv  f.  Hygiene,  Bd.  II. 
MIQUEL      ET      FREUDENREICH. — La       Semaine 

Mddicale. 

SEHLEN. — Fortschr.  d.  Med.,  Bd.  II,  S.  585. 
CHAMBERLAND. — Sur  un  Filtre  Dormant  de  1'Eau 

Physiologiquement     Pure.       Compt.    Rend.,  T. 

99,  p.  247. 
MOREAU     ET     PLANTYMAUSION. — La     Semaine 

Medicale. 
ANGUS  SMITH. — On  the  Examination  of  Waters. 

Rep.  to  the  Loc.  Gov.  Board. 

1885  CRAMER. — Die  Wasserversorgung  von  Zurich. 
CROOKSHANK. — Notes  from  a  Bact.  Labor.,  Lancet. 
BECKER. — Reichsmedicinalkalender. 

HESSE. — Ueber     Wasserfiltration.        Deut.     Med. 

Woch. 

SOYKA. — Prager  Med.  Woch. 
FRANKLAND. — Removal   of  Micro-organism   from 

Water.      Proc.  Roy.  Soc. 

1886  SOYKA. — Fortschr.  d.  Med. 

LAURENT. — Journal  de  Pharmacie  et  de  Chemie. 
WOLFFHUGEL  AND  RlEDEL. — Arbeit,  a.  d.  K.  Ges. 

Ain't 

HER^US. —  Zeitschr.  f.  Hygiene. 
C.  FRANKEL. — Zeitschr.  f.  Hygiene. 
MEADE  BOLTON. — Zeitschr.  f.  Hygiene. 
HESSE. — Zeitschr.  f.  Hygiene. 
BEUMER. — Deut.  Med.  Woch. 
FRANKLAND. — Proc.  Roy.  Soc. 
BlSGHOF. — Journ.  Soc.  Chem.  Industry. 
PFEIFFER. — Zeitschr.  f.  Hygiene. 


430 


APPENDIX. 


APPENDIX    G. 

TABLE  SHOWING  THE  MAGNIFYING  POWER  OF 
ZEISS'  OBJECTIVES. 


Ocular: 

a, 
a, 

a3 
a* 
aa 

A,  AA 

B,  BB 

C,  GC 

D,  DO 
E 

F 
G 
H 
J 
K 
L 


3 


7 

11 

15 

22 

12 

17 

24 

34 

20 

27 

38 

52 

4—12 

7—17 

10—24 

22 

30 

41 

56 

75 

38 

52 

71 

97 

130 

70 

95 

130 

175 

235 

120 

145 

195 

270 

360 

175 

230 

320 

435 

580 

270 

355 

490 

670 

890 

405 

540 

745 

1010 

1350 

260 

340 

470 

640 

8o5 

320 

430 

590 

805 

1075 

430 

570 

785 

1070 

1430 

570 

760 

1045 

1425 

1900 

770 

1030 

1415 

1930 

2570 

260 

340 

470 

640 

855 

380 

505 

695 

950 

1265 

605 

810 

1110 

1515 

2020 

a, 
a, 

a3 
a* 
aa 

A,  AA 

B,  BB 

C,  CC 

D,  DD 
E 

F 
G 
H 
J 
K 
L 
J 

A 
A 


INDEX. 


Abbe  condenser,  5 

Abrin,  312 

Abrus  precatorius,  312 

Abscesses,  197 

Acetate  of  potash,  16 

Acetic  acid,  9 

Achorion  Schoenleinii,  347 

Actinomyces,  334 

method  of  staining,  337 

Actinomycosis,  334 

Acute  infectious  osteomyelitis,  coccus 

of,  197 

Acute  yellow  atrophy,  214 
y^roniscopes,  364 
-^Eroscopes,  364 
Agar-Agar,  23 
peptone-broth,    preparation  of, 

21,  68 

Air,  examination  of,  361 
Aitken's  test  tube,  31,  93 
Alcohol,  7,  9 

acidulated,  9 

Alopecia  areata,  231 
Alum  carmine,  IO 
Ammonia,  10 
Aniline,  10 

—  water,  10 

Animals,  examination  of,  ill 
Anthrax,  281 
Antiseptics,  150 
Apparatus,  5 

ASCOCOCCUS,  222 

—  Billrothii,  122,  222 
.Ascomycetes,  347 
Asiatic  cholera,  255 
Aspergillus  albus,  349 

clavatus,  349 

flavus,  347 

fumigates,  347 

glaucus,  347 

niger,  347 

ochraceus,  349 

repens,  347 

Asphalte  lac,  16 

Attenuation  of  virus,  166,  234,  2^9 


B 

Babes',  method  of  examining  cultiva- 
tions, 50 

of  staining  comma  bacilli,  257 

of  staining  bacillus  of  leprosy, 

267 

Bacillus,  265 

acidi  lactici,  306 

aerophilus,  315 

alvei,  301 

amylobacter,  318 

anthracis,  281 

butyricus,  318 

caucasicus,  312 

cavicida,  238 

columbarum,  232 

—  cuniculicida,  235 
cyanogenus,  304 

—  dysodes,  313 

erythrosporus,  313 

figurans,  311 

Fitzianus,  306 

fluorescens,  303 

fcetidus,  315 

Hansenii,  313 

ianthinus,  303 

indicus,  240 

in  gangrenous  septicaemia,  271 

in  septicaemia  of  man,  271 

—  in  syphilis,  267 

in  tetanus,  301 

leprcc,  265 

liodermos,  244 

luteus,  241 

malariae,  270 

mallei,  292 

—  megaterium,  310 

mesentericus  fuscus,  316 

vulgatus,  316 

—  multipediculus,  244 

mycoides,  311 

Neapolitanus,  228 

cedematis  maligni,  293 

of  blue  milk,  304 

of  choleraic  diarrhoea  from  meat- 


poisoning,  270 


432 


INDEX. 


Bacillus,  of  diphtheria,  230 

of  glanders,  292 

of  jequirity,  312 

of  pneumo-enteritis  of  the  pig, 

299 

of  rhinoscleroma,  229 

of  septicaemia  of  mice,  297 

of  splenic  fever,  281 

of  swine-erysipelas,  300 

of  swine-typhoid,  299 

of  ulcerative    stomatitis    in  the 

calf,  298 

oxytocus  perniciosus,  238 

parvus  ovatus,  217 

pneumonicus  agilis,  237 

prodigiosus,  241 

pseudo-pneumonicus.  228 

putrificus  coli,  315 

• pyocyaneus,  303 

pyogenes  fostidus,  27  r 

ramosus  liquefaciens,  244 

—  saprogenes,  No.  I,  314 

—  No.  2,  314 
-No   3,  231,  314 

—  fcetidus,  315 

septicus,  314 

agrigenus,  234 

sputigenus,  236 

subtilis,  306 

trenmlus,  312 

tuberculosis,  272 

tumescens,  309 

typhosus,  268 

ureae,  243 

violaceus,  303 

virens,  117 

vitulorum,  231 

Bacteria,  chromogenic,  142 

classification  of,  175 

distribution  of,  148 

general  morphology  of,  117 

microscopical    examination    of, 

45 

pathogenic,  144 

saprogenic,  143 

zymogenic,  142 

Bacteriacese,  185,  224 
Bacteridie  du  charbon,  281 
Bacteridium  cyaneum,  218 
Bacterium,  225 

—  aceti,  243 

seruginosum,  303 

brunneum,  242 

cavicida,  238 

• chlorinum,  117 

cholerse  gallinarum,  232 

• coli  commune,  238 

crassum  sputigenum,  237 


Bacterium,  decalvans,  231 

fluorescens  liquefaciens,  242 

—  putidum,  242 

fcetidum,  315 

fusiforme,  246 

hyacinthi,  239 

ianthinum,  303 

indicum,  240 

in  diphtheria  of  calves,  231 

of  man,  230 

lactis  aerogenes.  239 

lineola,  248 

liodermos,  244 

litoreum.  246 

—  luteum.  741 

—  merismopedioides,  245 

multipediculum,  244 

navicula,  246 

Neapolitanum.  228 

—  of  Davaine's  septicaemia,  236 

—  of  diphtheria  of  pigeons,  232 
of  fowl-cholera,  232 

of  rhinoscleroma,  229 

of  septicaemia  in  rabbits,  235 

of  yellow  milk,  239 

oxytocum  perniciosum,  238 

Pasteuri,  236 

• Pasteurianum,  244 

Prlugeri,  246 

photometricum,  246 

pneumonias  cruposse,  225 

pneumonicum  agile,  237 

prodigiosum,  241 

pseudo-pneumonicum,  228 

ramosum  liquefaciens,  244 

rubescens,  325 

rubrum,  241 

saprogenes,  231 

septicum  agrigenum,  234 

sputigenum,  236 

syncyanum,  303 

synxanthum.  239 

termo,  248 

ureae.  242 

violaceum,  242 

—  viride,  117 

xanthinum,  239 

Zopfii,  244 

Balance  and  weights,  23 
Balmer-Frantzel  method,  279 
Baumgarten's  method,  278 
new  method,  278 

Bees,  bacillus  in  disease  of,  302 

Beggiatoa,  324 

methods  of  examining  species  of, 

324 

alba,  324 

mirabilis,  324 


INDEX. 


433 


Beggiatoa,  roseo-persicina,  324 
Bergamot  oil,  7 
Bibliography,  370 
Biere  malade.  207 
Bismarck-brown,  10 
Blackleg,  321 
Black  torula,  342 
Bleeding  host,  211 
Blood,  350 
—  rain,  241 

serum,  liquid,  90 

sterile,  29,  86 

Blue  milk,  bacillus  of,  304 

Borax  carmine,  10 

Botrytis  Bassiana,  350 

Bouillon,  89 

Bread-paste,  85 

Brush,  29 

Bulbed  tubes.  32 

Butyric  acid  fermentation,  bacillus  of, 


Camera-lucida,  17 
Canada-balsam,  17 
Caoutchouc  caps,  25 
Carbonate  of  soda,  24 
Carious  teeth,  220 
Cattle  plague,  204 
Cedar  oil,  6,  1 1 
Cell-contents,  119 
Cell- wall,  118 
Celloidin,  7,  55 
Cerebro-spinal  meningitis,  203 
Charbon  symptomatique,  320 
Chemical  composition,  117 

disinfectants.  153 

Cheshire's  trough,  71 
Chionyphe  Carteri,  350 
Cholera,  comma  bacillus  of,  250 

—  fowl,  233 

—  nostras,  comma  bacillus  in,  258 
Choleraic  diarrhoea  from  meat  poison- 
ing, bacillus  of,  270 

Cladothrix,  330 

dichotoma,  330 

— —  Fcersteri,  332 
Cladotricheae,  186,  330 
Classification,  175 
Flugge's.  1 80 

—  Hueppe's,  187 

Zopfs,  185 

Clostridium,  318 

—  butyricum,  318 

—  of  symptomatic  anthrax.  320 

polymyxa.  320 

Coccacese,  185 


Cocci,  methods  of  staining,  223 
Cohn-Mayer  fluid,  91 
Cohnia  roseo-persicina.  325 
Collection  of  water  samples,  367 
Comma-bacillus,  of  Finkler,  254 
—  of  Koch,  250 

in  cholera  nostras,  258 

Compressed  air,  140 

Cork,  7 

Cornil  and  Alvarez,  method  of,  230 

Cotton-wool,  23 

Cover-glass  impressions,  52 

preparations,  48 

—  double  coloration,  49 
Crenothrix,  322 

Kiihniana,  322 

Cutaneous  inoculation,  108 
Cutting  tissues,  53 


Damp-chambers,  26,  74 

Decalcifying  preparations,  54 

Dental  caries,  203 

Desiccator,  43 

Diphtheria,  202.  230 

Diplococcus  albicans  tardissimus,  210 

Disinfectants,  150 

Dissecting  boards,  41 

—  case,  41 
Dissection,  ill 
Distribution  of  bacteria,  148 
Double  coloration.  49 
Double-stain  spore-bearing  bacilli,  291 
Double-staining,  51 
Drawing  apparatus,  17 
Dressing-case,  41 
Drinking  water.  322 
Drop-cultures.  94 
Drop-culture  slides,  32 


Ebner's  solution,  7 

Ehrlich's  method  of  examining  culti- 
vations, 50,  276 

and  eosin,  280 

Electricity,  141 

—  application  of,  104 
Embedding  tissues,  55 
Empusa  muscle,  343 

—  radicans,  341 
Endocarditis  ulcerosa,  2O2 
Eosin,  II 

Erysipelas,  2OO 

malignum,  bacillus  des,  300 

Ether,  II 

28 


434 


INDEX. 


Eurotium  aspergillus  glaucus,  347 

aspergillus  niger,  348 

repens,  347 

Examination,  of  plate  cultivations,  78 

—  of  test-tube  cultivations,  71 
Experiments  on  living  animals,  107 


Farrant's  solution,  17 
Favus,  347 
Filter,  making,  66 

paper,  24 

Fire  blight,  218 

Flagella,  to  stain,  52 

Flagellated  protozoa  in  blood,  350 

Flagellum,  128 

Flannel,  24 

Flecksucht,  239 

Folded  filter,  67 

Foot  and  mouth  disease,  205 

Form,  122 

Foulbrood,  302 

Fowl-cholera,  bacterium  of,  232 

Frankel's  method,  279 

Friedlander,  method  of,  227 

Frogspawn  fungus,  262 

Fuchsine,  1 1 


Gangrene,  214,  272 
Gas,  burners,  35,  36 

—  chambers,  103 

pressure  regulator,  Moitessier's,37 

Gases,  effect  of,  140 
Gattine,  239 
Gelatine,  8,  23 

—  peptone   broth,   preparation   of, 

21,  64 

; —  plates,  76 
Gelatinous  envelope,  120 
Gentian  violet,  1 1 
Gibbes',  first  method,  277 
magenta  solution,  13 

—  new  method,  277 

—  solution  for  double-staining,  1 1 
Glanders,  bacillus  of,  292 

Glass,  bells,  26 

benches,  27 

capsules,  31 

dishes,  26,  28 

jar,  29 

plates,  27 

rods,  27 

vessels,  22 

Glycerine,  gelatine,  8,  57 
gum,  17 


Glycerine,  pure,  12 
Gomme  de  sucrerie,  262 
Gonorrhoea,  208 
Gram's,  method  of  staining,  59 

solution,  12 

Growth,  circumstances  affecting,  139 

products  of,  141 

Gum,  8 


H 

Haematomonas  carassii,  355 
-  cobitis,  353,  355 

Evansi,  357 

Haematoxylin  solution,  12 
Haemophilia  neonatomm,  214 
Hardening  preparations,  54 
Hay-bacillus,  306 

methods  of  staining,  309 

Heat  regulator  (Meyer's),  40 

(Reichert's),  37 

(Schlosing's),  34     . 

Herpes  tonsurans,  347 
Hesse's  apparatus.  363 
His'  method  of  examining  cultivations, 

51 

Hollis'  glue,  17 
Hot  air  and  steam,  159 
Hot-air  steriliser,  20 
Hot- water  filter,  21 
Hydrophobia,  215 
Hyphomycetes,  342 
Hypodermii,  343 


I 

Immunity.  162 
Impression-preparations,  52 
Incubators,  32 

—  Babes',  36 

—  D' Arson val's,  32 
Inoculating  test-tubes,  69,  74 

cutaneous  and  subcutaneous,  108 

of  potatoes,  83 

protective,  163 

Instruments,  41 

Intermittent  fever,  bacillus  of,  177 

Iodine  solution,  12 

--  (Gram),  12 
Iron  box,  26 

Isolation  of  micro-organisms,  114 
Israel's,  case,  28 
warming  apparatus,  101 

J 

Japanese  isinglass,  23 
Jequirity,  bacillus  of.  312 


INDEX. 


435 


K 

Kaatzer's  method,  280 

Kephir,  313 

Kleinenberg's  solution,  8 

Koch's  apparatus  for  examination  of 
air,  362 

— —  method     for     staining    comma- 
bacilli,  257 

method  of  plate-cultivation,  72 

original  method  for  staining  the 

tubercle  bacillus,  275 

postulates,  27 

solution  (methylene-blue),  13 

—  solution  (methyl- violet),  14 


Laboratory  requisites,  42 
Lactic  acid,  24 
Leprosy,  266 
Leptothrix,  329 

—  buccalis,  329 

—  gigantea,  330 

—  ochracea,  331 
—  parasitica,  331 

Leptotricheae,  185,  321 
Leuconostoc,  262 

—  mesenteroides,  262 
Leukaemia,  197 
Levelling  apparatus,  73 
Lichtheim's  method",  279 
Light,  141 

Liquid  media,  preparation  of,  31,  8l 
Lister's  flasks,  31,  92 
Lithium-carmine  solution,  12 
Litmus  papers,  23 
Living  animal,  107 
Lochial  discharges,  210 
Loffler's  solution,  13 
Lustgarten,  method  of,  267 

M 

Madura-foot,  350 

Magenta  solution  (Gibbes').  13 

Malaria,  270 

Malignant- oedema,  bacillus  of,  293 

—  pustule,  281 
Marsh-spirochaete,  260 
Measles,  214 
Merismopedia,  208 

—  gonorrhoea,  208,  223 
Methylene-blue,  II,  13 
Methyl-violet,  13 

Meyer's  thermo-regulator,  40 

Micrococcus,  213 

— —  albicans  amplus,  2IO 

—  amylivorus,  218 


Micrococcus,  aurantiacus,  218 
candicans,  219 

—  candidus,  219 
chlorinus,  218 

cholerae  gallinarum,  232 

cinnabareus,  220 

citreus-conglomeratus,  209 

coronatus,  207 

crepusculum,  22O 

cyaneus,  218 

—  endocarditicus,  192 
flavus  desidens,  208 

liquefaciens,  220 

tardigradus,  220 

foetidus,  220 

fulvUS,  221 

—  hsematodes,  219 
indicus,  240 

—  in  acute  yellow  atrophy,  214 
in  gangrene,  214 

in  haemophilia  neonatorum,  214 

in  measles,  214 

in  rabies,  215 

—  in  scarlatina,  213 

—  in  typhus,  214 

in  whooping  cough,  214 

lacteus  faviformis,  221 

luteus,  219 

of  foot  and  mouth  disease,  205 

of   progressive    suppuration    in 

rabbits,  216 

of  pyaemia  in  mice,  217 

of  pyaemia  in  rabbits.  216 

of  septicaemia  in  rabbits,  215,  224 

—  ovatus,  239 

—  parvus,  217 
— r—  prodigiosus,  241 

—  pyocyaneus,  303 

pyogenes  tenuis,  215 

radiatus,  208 

rosaceus,  219 

subflavus,  210 

tetragonus,  209,  224 

ureae,  242 

—  versicolor.  221 

vio'aceus,  219 

viticulosus,  221 

Micro-organisms,    causa    causans    of 

diseases,  2 

inhalation  of,  107 

—  isolation  of,  114 
Micro-photographic  apparatus,  17 
Microscope,  5 
Microsporon  furfur,  347 
Microtome,  6 
freezing,  7 


Microzyma  bombycis,  207 
Microzyme  test,  366 


436 


INDEX. 


Mikrokokken  in  faulenden  Substraten, 

220 

Milk,  90,  238,  239,  304,  306 
Miquel's  bulbs,  94 
Mi'ist-chambers,  96 
Monas,  crepusculum,  220 
hsemorrhagicum,  214 

—  Okenii,  327 

vinosa,  327 

Warmingii,  327 

Morphology,  117 
Moulds,  340 
Mouse-cages,  41 
Movement,  124 

effect  of,  140 

Mucor,  aspergillus,  346 
corymbifer,  346 

—  fusiger,  346 

—  macrocarpus,  346 
melittophorus,  346 

mucedo,  345 

phycomyces,  346 

racemosus,  345 

rhizopodiformis,  346 

—  stolonifer,  346 
Muller's  fluid,  8 

Mycoderma  cerevisiae  et  vini,  341 
Myconostoc  gregarium,  333 


N 

Nature  of  the  soil,  139 
Neelsen's  method,  278 

—  solution,  14 

Nicati  and  Rietsch's  method,  257 
Nitric  acid,  14 
Nosema  bombycis,  239 
Nutrient,  agar-agar,  68 

—  gelatine,  64 

—  media,  62 
Nutrition,  136 


CEdema,  293 

Oidium,  albicans,  341,  347 
-  lactis,  347 

;  Tuckeri,  347 

Ophidomonas  sanguinea,  327 
Orseille  (Wedl),  14 
Orth's  method,  61 

modification  of  Ehrlich's  method, 

277 

solution,  12,  ic 

Ose,  25 
Osmic  acid,  8 
Osteomyelitis,  197 


Page's  thermo-regulator,  39 
Panhistophyton  ovatum,  239 
Paper  trays,  9 
Paraffine,  9 
Parrot  disease,  206 
Paste-cultivations,  85 
Pasteur's  apparatus,  93 

fluid,  91 

septicaemia,  293 

Peach-coloured  bacterium,  325 
Pebrine,  239 

Penicillium  glaucum,  349 
Peptonum  siccum,  23 
Perlsucht,  275 
Peronospora  infestans,  344 
Peters'  method,  279 
Pfuhl-Petri's  method,  280 
Phosphorescence,  246 
Photo-micrographic  apparatus,  17 
Phragmidiothrix,  328 

multiseptata,  328 

Phycomycetes,  344 
Picric  acid,  14 
Picro-carmine  (Ranvier),  15 

lithium-carmine  (Orth),  15 

Pilobolus,  345 
Pink  torula,  342 
Pipette,  29 

Pityriasis  versicolor,  347 
Plate-cultivations,  72,  367 
Plate-glass,  26 
Platinum  needles,  25 
Plaut's  method,  337 
Pleomorphism,  182 
Pleuritis,  228 
Pneumococcus,  226 
Pneumo-enteritis  of  the  pig,  299 
Pneumonia,  227 

method  of  staining  bacteria  of, 

227 

Potash  solution,  15 
Potato-bacillus,  316 

bacterium,  244 

—  cultivations,  77 
Potatoes,  disease  of,  226 
Potato-knives,  28 

paste,  84 

Pouchet's  aeroscope,  364 

Pravaz'  syringe  (Koch's  modification), 

45 

Protected  burners,  35 
Protective  inoculation,  163 
Proteus,  mirabilis,  247 

vulgaris,  246 

Zenkeri,  248 

Ptomaines,  144 


INDEX. 


437 


Puerperal  fever,  198,  201 
Pus,  195.  197,  199.217,  271 
Putrefaction,  247 
Pyaemia,  195,  198 

in  mice,  217 

4 in  rabbits,  216 

Pyocyanin,  303 


Quarter-evil,  321 


R 


Rabies,  215 

Ranvier's  picro-carmine,  15 

Rauschbrand.  320 

Ray-fungus,  334 

Re-agents,  7,  9,  16 

Reichert's  therm o- regulator,  37 

Relapsing  fever,  spirillum  of,  249 

Reproduction,  128 

Respiration,  136 

Rhabdomonas  rosea,  328 

Rhinoscleroma,  230 

Rindfleisch's  method,  276 

Rosaniline-hydrochlorate,  1 1 

Rothlauf,  300 

Rouget  du  pore,  204,  300 


Saccharomyces,  albicans,  341 

—  apiculatus,  341 

—  cerevisise,  340 

conglomeratus,  341 

• ellipsoid eus,  340 

exiguus,  341 

glutinis,  342 

mycoderma,  341 

—  niger,  342 

pastorianus,  341 

ro?aceus,  342 

sphsericus,  341 

Saccharomycetes,  340 
Safety  burner,  Koch's,  36 
Safranine,  15 

Salmon,  disease  of,  344 
Salt  solution,  15 
Saprolegnia,  344 
Sarcina,  210 

—  alba,  212 

aurantiaca,  21 1 

hyalina,  212 

intestinalis,  212 

litoralis,  212 

lutea,  210 

Reitenbachii,  212 

— —  urinae,  212 


Sarcina,  ventriculi,  21 1 
Scalpels,  29 
Scarlatina,  214 
Schizomycetes,  180,  185,  194 
Schlosing's  membrane-regulator,  34 
Schiitz,  method  of,  293 
Senkewitsch's  method,  280 
Septicaemia,  bacillus  of  gangrenous, 
272 

bacterium  of,  235,  236 

consecutive  to  anthrax,  205 

in  rabbits,  215 

of  man,  271 

of  mice,  297 

Serum-inspissator,  30 

steriliser,  29 

Silkworms,  207,  239 
Siphon-apparatus,  42 
Soil,  examination  of,  365 
Spermaceti,  9 
Sphaerotilus  natans,  333 
Spirillum,  249 

attenuatum,  262 

cholerse  Asiaticae,  250 

Finklerii,  258 

—  leucomelaneum,  262 

method  of  staining,  250 

Obermeieri,  249 

—  of  relapsing  fever,  249 

plicatile,  260 

rosaceum,  327 

Rosenbergii,  262 

sanguineum,  327 

—  serpens,  261 

—  sputigenum,  259 

—  tenue,  261 

tyrogenum,  260 

undula,  261 

violaceum,  327 

volutans,  261 

Spirit-level,  26 
Spirochaete  buccalis,  329 

Obermeieri,  249 

Spiromonas  Cohnii,  333 

—  volubilis,  333 
Splenic  fever,  281 

Spores,  method  of  staining,  51 
*  preservation  of,  286 

Sputum,  221 

septicaemia.  236 

Staining  bacteria,  57 

methods  of,  223 

—  actinomyces,  337 

bacilli  of  ulcerative  stoma- 
titis, 299 

bacillus,  anthracis,  290 

of    butyric   acid    fer- 
mentation, 320 


433 


INDEX. 


Staining        methods,       bacillus,      of 
glanders,  293 

of  leprosy,  266 

of  rhinoscleroma,  230 

—  of  syphilis,  267 

of  typhoid  fever,  269 

bacteria  in  diphtheria,  231 

of  pneumonia,  227 

—  comma-bacilli,  257 

—  hay  bacillus,  309 

leptothrix  buccalis,  329 

spirillum  Obermeieri,  250 

tubercle  bacilli,  275 

Staphylococcus  cereus  albus,  199 
cereus  flavus,  200 

—  pyogenes  albus,  198 
aureus,  197 

—  citreus,  199 
Steam,  159 

—  steriliser,  19 
Sterilisation,  apparatus,  19 

—  of  glass  plates,  73 
Sternberg's  bulbs,  31,  92 
Streptococcus,  195 

—  articulorum,  202 
bombycis,  207 

—  cereus  albus.  199 

flavus,  200 

— —  coronatus,  207 

— : —  erysipelatosus,  200 
flavus  desidens.  208 

—  in  cerebro-spinal  meningitis,  203 

—  in  dental  caries,  203 
in  diphtheria,  202 

—  in  endocarditis,  201 

—  in  puerperal  fever,  20 1 

insectorum,  207 

in  yellow  fever,  203 

of  cattle  plague,  204 

•  -  of  foot  and  mouth  disease,  205 

—  of  progressive  tissue  necrosis  in 

mice,  205 
of    septicaemia    consecutive    to 

anthrax,  205 

of  swine  erysipelas,  204 

perniciosus,  206 

—  pyogenes,  195 

—  albus,  198 
aureus,  197 

citreus,  199 

malignus,  197 

—  radiatus,  208 

septicus,  205 

toxicatus,  201 

—  variolae  et  vaccinise.  203 

viscosus,  207 

Streptothrix  Fcersteri,  332 
Sulphuric  acid,  15 


Surra,  352 
Sweat,  human,  219 
Swine-erysipelas,  204,  217,  300.  315 

typhoid,  299 

Symptomatic  anthrax,  320 
Syphilis,  267 


Table  salt,  23 

Tarichium  megaspermum.  344 
Teeth,  carious,  220,  259 
Temperature,  effect  of,  139 
Test-tube  cultivations,  69,  369 

stands,  24 

water-bath,  21 

Tetanus.  301 
Thermometers,  27 
Thermo-regulator,  38,  39,  40 
Tilletia  caries,  343 
Torula  cerevisise,  340 
Transport  of  water  samples,  367 
Trichomonas  sanguinis,  360 
Tricophyton  tonsurans,  347 
Tripod  levelling-stand,  25 
Tuberculosis,  bacillus  of,  272 
Turpentine,  15 
Typhoid  fever,  269 
Typhus,  214 


U 

Ulcerative  stomatitis  in  the  calf,  298 
Ureihral  pus.  210 
Urine,  90,  243 
Urocystis  occulta,  343 
Ustilago  carbo,  343 


Vaccinia,  203 

Vaginal  secretions,  210,  221 
Valentin's  knife,  7 
Variola,  203 
Vaseline,  32 
Vegetables,  86 
Vegetable  infusions,  90 
Vesuvin,  15 
Vibrio,  317 

—  rugula,  317 

—  serpens,  261 
Vin  filant,  207 


W 

Warm  stages,  97 
Warming  apparatus,  101 
Water,  16 


INDEX. 


439 


Water,  bath,  21 

—  examination  of,  366 
Wedl's  orseille,  14 
Weigert-Ehrlich  method  of  staining, 

61 

Weigert's  method  of  staining,  58,  291 
Whooping  cough,  214 
Wire  cages,  24 
Wooden- tongue,  335 
Woolsorter's  disease,  281 
\Vurzel-bacillus,  311 


Xylol,  9,  19 

Yeast  fungi,  340 
Yeasts,  340 
Yellow  fever,  203 


Ziehl's  method,  279 
Zinc- white,  17 


DATE  DUE  SLIP 

UNIVERSITY  OF  CALIFORNIA  MEDICAL  SCHOOL  LIBRARY 

THIS  BOOK  IS  DUE  ON  THE  LAST  DATE 
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1887     2d 


shank,  E.M.   13561 
Mahual  of  bact sriology, 


